Compositions, kits and methods for detection of campylobacter nucleic acid

ABSTRACT

The disclosed invention is related to compositions, kits and methods comprising one or more oligomers targeting 16S rRNA target nucleic acid from  Campylobacter  species  jejuni, coli  and/or  lari . Compositions include amplification oligomers, detection probe oligomers and/or target capture oligomers. Kits and methods comprise at least one of these oligomers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication No. 61/114,547, filed on Nov. 14, 2008, which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to detection of the presence of Campylobacterjejuni (C. jejuni), Campylobacter lari (C. lari), or Campylobacter coli(C. coli) bacteria in a sample by using molecular biological methods,and specifically relates to detection of C. jejuni, C. lari, or C. coliin a sample by amplifying nucleic acids from C. jejuni, C. lari, or C.coli and detecting the amplified nucleic acid sequences.

BACKGROUND

Campylobacter contamination is one of the major causes of food-borneillness, and can lead to Campylobacteriosis. Campylobacter are also amajor cause of diarrhoeal illness in humans and are generally regardedas the most common bacterial cause of gastroenteritis worldwide. In theUnited States, Campylobacter are estimated to affect two million peoplea year. Common routes of transmission are ingestion of contaminatedfood, water or milk, eating raw meat, person-to-person sexual contact,and fecal-oral. The infectious dose for Campylobacter may be as low as400-500 bacteria as shown by human feeding studies, however, theinfectious dose and the dose response are dependent upon the strainsused, and the age and physical condition of the individuals. Symptomsappear 2-10 days after ingesting the bacteria and include fever,abdominal cramps, and mild to severe diarrhea, which may be bloody. Mostinfections are caused by C. jejuni, normally found in cattle, swine, andbirds, where it is non-pathogenic, however, the illness may also becaused by C. coli (also found in cattle, swine, and birds) and C. lari(present in seabirds in particular). Some strains of C. jejuni produce acholera-like enterotoxin, which is important in the watery diarrheaobserved in infections. Most Campylobacter infections clear on their ownor with the aid of antibiotics, however, long-term sequelae includereactive arthritis, Guillain-Barré syndrome and Miller Fisher Syndromes.

Studies have shown that Campylobacter can be isolated from a widevariety of sources, including chicken, cattle, pets, goats, sheep andpond water and river water. Milk, particularly unpasteurised (raw) milk,and poultry are the most common food items associated with Campylobacterinfection. Campylobacter may also be isolated from red meat, but thefrequency of contamination is generally much lower than poultry.Isolation of Campylobacter from food sources is difficult because thebacteria are usually present in very low numbers since they generally donot multiply in the foods they infect. Culture methods require anenrichment broth containing antibiotics, special antibiotic-containingplates, incubation at two different temperatures, and a microaerophilicatmosphere with an elevated concentration of carbon dioxide. Isolationcan take several days to a week. Identification of Campylobacter usingcurrent USDA methods for detection can take 2-3 days. There is a needfor a rapid, sensitive and accurate method to detect C. jejuni, C. lari,or C. coli so that the contaminating or infectious source can beaccurately detected and eliminated. There is also a need for methodsthat allow rapid and accurate diagnosis of C. jejuni, C. lari, or C.coli infections in humans so that infected individuals may be treatedpromptly to limit morbidity and prevent further infections.

SUMMARY

The present invention relates to compositions, kits, and methods used inthe detection of common pathogenic strains of Campylobacter,particularly, Campylobacter jejuni, Campylobacter coli, andCampylobacter lari. The invention is based at least in part on thediscovery that certain Campylobacter sequences are surprisinglyefficacious for the detection of Campylobacter. In certain aspects andembodiments, particular regions of the Campylobacter 16S rRNA have beenidentified as preferred targets for nucleic acid amplificationreactions, which provide improvements in relation to specificity,sensitivity, or speed of detection as well as other advantages.

Therefore, according to one embodiment, there are provided compositionsfor use in a Campylobacter nucleic acid amplification assay, wherein thecompositions are amplification oligomers for amplifying a Campylobactertarget nucleic acid. In certain aspects, the compositions include a T7Provider oligonucleotide and a primer oligonucleotide, both of which arecapable of stably hybridizing to a C. jejuni target nucleic acid and aC. coli, a C. lari or a C. coli and a C. lari target nucleic acid; inwhich the T7 Provider oligonucleotide is configured to target a sequencein a region of Campylobacter nucleic acid corresponding to bases fromabout 14-150 of GenBank Accession No.: AF393202.1, gi:20378208 16S rRNA(SEQ ID NO:91) and the primer oligonucleotide is configured to target asequence in a region of Campylobacter nucleic acid. Ordinarily skilledartisans will understand that one of the amplification oligomers isconfigured sense and the other antisense to the reference sequence inorder that the amplification oligomers can be used in an amplificationassay.

In another embodiment, there are provided kits that include thecompositions provided herein. In certain aspects, the kits include a T7Provider oligonucleotide and a primer oligonucleotide, both of which arecapable of stably hybridizing to a C. jejuni target nucleic acid and aC. coli, a C. lari or a C. coli and a C. lari target nucleic acid, inwhich the T7 Provider oligonucleotide is configured to target a sequencein a region of Campylobacter nucleic acid corresponding to bases fromabout 14-150 of GenBank Accession No.: AF393202.1, gi:20378208 16S rRNAand the primer oligonucleotide is configured to target a sequence in aregion of Campylobacter nucleic acid. Ordinarily skilled artisans willunderstand that one of the amplification oligomers is configured senseand the other antisense to the reference sequence in order that theamplification oligomers can be used in an amplification assay.

In another embodiment, there are provided methods for detecting thepresence of Campylobacter in a sample using the compositions and/or kitsprovided herein. In certain aspects, the methods use a T7 Provideroligonucleotide and a primer oligonucleotide that stably hybridizing toa C. jejuni target nucleic acid and a C. coli, a C. lari or a C. coliand a C. lari target nucleic acid, and in which the T7 Provideroligonucleotide is configured to target a sequence in a region ofCampylobacter nucleic acid corresponding to bases from about 14-150 ofGenBank Accession No.: AF393202.1, gi:20378208 16S rRNA and the primeroligonucleotide is configured to target a sequence in a region ofCampylobacter nucleic acid. Ordinarily skilled artisans will understandthat one of the amplification oligomers is configured sense and theother antisense to the reference sequence in order that theamplification oligomers can be used in an amplification assay.

In one aspect, the T7 Provider is configured to target a sequence in aregion of Campylobacter nucleic acid corresponding to bases 83-150 ofGenBank Accession No.: AF393202.1, gi:20378208. In another aspect, theT7 Provider is configured to target a sequence in a region ofCampylobacter nucleic acid corresponding to bases 108-150 of GenBankAccession No.: AF393202.1, gi:20378208. In another aspect, the T7Provider is configured to target a sequence in a region of Campylobacternucleic acid corresponding to bases 115-150 of GenBank Accession No.:AF393202.1, gi:20378208. In another aspect, the T7 Provider isconfigured to target a sequence in a region of Campylobacter nucleicacid corresponding to bases 115-135 of GenBank Accession No.:AF393202.1, gi:20378208. In another aspect, the T7 Provider isconfigured to target a sequence in a region of Campylobacter nucleicacid corresponding to bases 125-150 of GenBank Accession No.:AF393202.1, gi:20378208.

In one aspect, the primer oligonucleotide is configured to target asequence in a region of Campylobacter nucleic acid corresponding tobases from about 62-226 of GenBank Accession No.: AF393202.1,gi:20378208 (SEQ ID NO:91). In another aspect, the primeroligonucleotide is configured to target a sequence in a region ofCampylobacter nucleic acid corresponding to bases from about 170-226 ofGenBank Accession No.: AF393202.1, gi:20378208. In another aspect, theprimer oligonucleotide is configured to target a sequence in a region ofCampylobacter nucleic acid corresponding to bases from about 170-212 ofGenBank Accession No.: AF393202.1, gi:20378208. In another aspect, theprimer oligonucleotide is configured to target a sequence in a region ofCampylobacter nucleic acid corresponding to bases 184-212 of GenBankAccession No.: AF393202.1, gi:20378208. In another aspect, the primeroligonucleotide is configured to target a sequence in a region ofCampylobacter nucleic acid corresponding to bases 170-205 of GenBankAccession No.: AF393202.1, gi:20378208.

In one aspect, the T7 Provider is selected from the sequences of SEQ IDNOS:18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 84,85, 86, 87, 88 and their complements. In another aspect, the T7 Providercomprises a target hybridization sequence that is selected from thesequences of SEQ ID NOS:19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, and their complements; and further comprises a promoternucleotide sequence selected from the group consisting of; atranscriptional promoter recognized by an RNA Polymerase, atranscriptional promoter recognized by a bacteriophage T7 RNApolymerase, SEQ ID NO:75 and SEQ ID NO:76. In one aspect, the primeroligonucleotide is selected from the sequences of SEQ ID NOS: 1-17 andtheir complements.

In one preferred embodiment, the T7 Provider is selected from thesequences of SEQ ID NOS:18, 20, 26, 28, 30, 32 and 48. In otherpreferred embodiments, the primer oligonucleotide selected from thesequences of SEQ ID NOS:1, 4, 7, 8, 10, 12. In one preferred embodiment,the T7 Provider has the sequence of SEQ ID NO:26 and the primeroligonucleotide has the sequence of SEQ ID NO:7. In one preferredembodiment, the T7 Provider has the sequence of SEQ ID NO:32 and theprimer oligonucleotide has the sequence of SEQ ID NO:8.

In certain embodiments of the compositions, methods and kits, a T7Provider oligonucleotide is configured to comprise a target hybridizingsequence that is 21-32 nucleotides in length and that is at least 70%;or 75%; or 80%; or 85%; or 90%; or 100% complementary to a sequence in aregion of Campylobacter nucleic acid corresponding to bases 83-150 ofGenBank Accession No.: AF393202.1, gi:20378208. In certain embodimentsof the compositions, methods and kits, a T7 Provider oligonucleotide isconfigured to comprise a target hybridizing sequence that is 21-32nucleotides in length and that are complementary to a sequence in aregion of Campylobacter nucleic acid corresponding to bases 83-150 ofGenBank Accession No.: AF393202.1, gi:20378208, but has 1 mismatch; or 2mismatches; or 3 mismatches; or 5 mismatches as compared to the portionof GenBank Accession No.: AF393202.1, gi:20378208 to which it iscomplementary. The T7 Provider oligonucleotide of these certainembodiments further comprises a promoter nucleotide sequence selectedfrom the group consisting of; a transcriptional promoter recognized byan RNA Polymerase, a transcriptional promoter recognized by abacteriophage T7 RNA polymerase, SEQ ID NO:75 and SEQ ID NO:76.

In certain embodiments of the compositions, kits and methods, a primeroligonucleotide is configured to comprise a target hybridizing sequencethat is 19-35 nucleotides in length and that is at least 70%; or 75%; or80%; or 85%; or 90% or 100% identical to a sequence in a region ofCampylobacter nucleic acid corresponding to bases from about 170-226 ofGenBank Accession No.: AF393202.1, gi:20378208. In certain embodiments,a primer oligonucleotide is configured to comprise a target hybridizingsequence that is 19-35 nucleotides in length and that is identical to asequence in a region of Campylobacter nucleic acid corresponding tobases from about 170-226 of GenBank Accession No.: AF393202.1,gi:20378208 but has 1 mismatch; or 2 mismatches; or 3 mismatches; or 5mismatches as compared to the portion of GenBank Accession No.:AF393202.1, gi:20378208 to which it is complementary.

In one embodiment, one or more additional oligonucleotide types and/orother amplification reagents that serve to facilitate or improve one ormore aspects of the amplification reaction may be included. For example,in addition to a T7 Provider and/or a primer oligonucleotide, additionaloligonucleotides may further include one or more of: a detectionoligonucleotide, a blocker oligonucleotide, a target captureoligonucleotide, and the like.

In one embodiment, the compositions, kits, and/or methods may furtherinclude or use a detection oligonucleotide, preferably a linear orhairpin detection oligonucleotide, more preferably a torcholigonucleotide or molecular beacon oligonucleotide. In one aspect, thedetection oligonucleotide stably hybridizes to an amplification productgenerated from a Campylobacter target nucleic acid using at least oneamplification oligomer described herein, and more preferably using twoamplification oligomers described herein. In one aspect, the detectionoligonucleotide stably hybridizes to amplification products generatedfrom a C. jejuni target nucleic acid and a C. coli, a C. lari or a C.coli and a C. lari target nucleic acid using at least one amplificationoligomer described herein, and more preferably using two amplificationoligomers described herein. In one aspect, the detection oligonucleotideis from 10 to 70 nucleotides in length and stably hybridizes to the “+”or “−” strand of a region of SEQ ID NO:91, or RNA equivalent thereof. Inone aspect, the detection oligonucleotide is a torch oligonucleotideselected from the sequences of SEQ ID NOS:59-70 and their complements.In one aspect, the detection oligonucleotide is a torch oligonucleotideselected from the sequences of SEQ ID NOS:62, 63, 65, 66, 67, and 68,and their complements. In one aspect, the detection oligonucleotide is atorch oligonucleotide selected from the sequences of SEQ ID NO:68, SEQID NO:67, and their complements. In one aspect, the detectionoligonucleotide is a torch oligonucleotide having the sequence of SEQ IDNO:67 or its complement.

In one embodiment, the compositions, kits, and/or methods may furtherinclude or use a blocker oligonucleotide. In one aspect, the blockeroligonucleotide is selected from the sequences of SEQ ID NOS:50-57 andtheir complements. In one aspect, the blocker oligonucleotide isselected from the sequences of SEQ ID NOS:50, 53 and 56, and theircomplements. In one aspect, the blocker oligonucleotide has the sequenceof SEQ ID NOs:50 or its complement.

In one embodiment, the compositions, kits, and/or methods might furtherinclude or use a target capture oligonucleotide. In one aspect, thetarget capture oligonucleotide is selected from the sequences of SEQ IDNOS: 71, 73, and their complements. In one aspect, the target captureoligonucleotide has the sequence of SEQ ID NOs:73 or its complement.

In some aspects, the compositions are used in a transcription-mediatedamplification assay (hereinafter “TMA”). In some aspects, there areprovided kits for performing a transcription-mediated amplificationassay. In some aspects, there are provided methods for performing atranscription-mediated amplification assay. In certain embodiments, thecompositions, kits, and/or methods may include or use one or moreoligonucleotides such as a: T7 Provider, primer oligonucleotide,detection oligonucleotide, blocker oligonucleotide, Torcholigonucleotide, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows nucleotide residues 1-450 from the 16S rRNA nucleotidesequence of Campylobacter jejuni GenBank Accession Number AF393202.1,GI:20378208 (May 1, 2002).

FIG. 2 shows the nucleotide sequence of the 16S rRNA nucleotide sequenceof Campylobacter jejuni GenBank Accession Number AF393202.1, GI:20378208(May 1, 2002), which is also SEQ ID NO:91.

DETAILED DESCRIPTION

Disclosed are compositions, kits and methods for amplifying anddetecting one or more of C. jejuni, C. lari, or C. coli target nucleicacid present in a sample. These target nucleic acids are 16S rRNA and/orgenes encoding 16S rRNA from one or more of C. jejuni, C. lari, or C.coli. Preferably, the samples arc food samples, water samples,industrial samples, environmental samples or biological samples. Thecompositions, kits and methods provide oligonucleotide sequences thatspecifically recognize these target nucleic acids. Such oligonucleotidesmay be used as amplification oligonucleotides, which may includeprimers, promoter primers, blocked oligonucleotides, and promoterprovider oligonucleotides, whose functions have been describedpreviously (e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159;5,399,491; 5,554,516; 5,824,518; and 7,374,885). Other oligonucleotidesmay be used as probes for detecting amplified sequences of C. jejuni, C.lari, and/or C. coli.

The amplification step includes contacting the sample with one or moreamplification oligonucleotides specific for a target sequence within thetarget nucleic acid. Amplification synthesizes additional copies of thetarget sequence or its complement by using at least one nucleic acidpolymerase. Depending on the amplification method used and theconfiguration of the amplification oligomer, amplification synthesizes acomplementary DNA or RNA sequence corresponding to the target sequenceor its complement. Ordinarily skilled artisans understand theamplification process employed in the various amplification techniques,and understand that the amplification product strands are sense orantisense to the template strand and may be RNA or DNA strands. Oneembodiment for detecting the amplified product uses a hybridizing stepthat includes contacting the amplified product with at least one probespecific for a portion of the amplification product. Some otherdetection methods include, gel electrophoresis, dye staining,radiolabeling and mass spectrometry, to name a few.

A detecting step may be performed after the amplification reaction iscompleted, or may be performed simultaneous with amplifying the targetregion, e.g., in real time. In one embodiment, the detection step allowshomogeneous detection, e.g., detection of the hybridized probe withoutremoval of unhybridized probe from the mixture (e.g., U.S. Pat. Nos.5,639,604 and 5,283,174). In embodiments that detect the amplifiedproduct near or at the end of the amplification step, a linear probe maybe used to provide a signal to indicate hybridization of the probe tothe amplified product. In other embodiments that use real-timedetection, the probe may be a linear probe, such as a dual labeledTaqMan probe detected upon 5′->3′ exonuclease degradation of the probe,or may be a hairpin probe, such as a molecular beacon, molecular torch,or hybridization switch probe labeled with a reporter moiety that isdetected when the hairpin probe binds to amplified product. Variousforms of such probes have been described previously (e.g., U.S. Pat.Nos. 5,118,801; 5,210,015; 5,312,728; 5,538,848; 5,925,517; 6,150,097;6,849,412; 6,835,542; 6,534,274; and 6,361,945; and US Pub. Nos.20060068417A1; and US Pub. No. 20060194240A1).

To aid in understanding aspects of the disclosure, some terms usedherein are described in more detail. All other scientific and technicalterms used herein have the same meaning as commonly understood by thoseskilled in the relevant art, such as may be provided in Dictionary ofMicrobiology and Molecular Biology, 2nd ed. (Singleton et al., 1994,John Wiley & Sons, New York, N.Y.), The Harper Collins Dictionary ofBiology (Hale & Marham, 1991, Harper Perennial, New York, N.Y.), andreferences cited herein. Unless mentioned otherwise, the techniquesemployed or contemplated herein are standard methods well known to aperson of ordinary skill in the art of molecular biology.

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “a nucleic acid,” is understood torepresent one or more nucleic acids. As such, the terms “a” (or “an”),“one or more,” and “at least one” can be used interchangeably herein.

“Sample” includes any specimen that may contain C. jejuni, C. lari,and/or C. coli or components thereof, such as nucleic acids or fragmentsof nucleic acids. Samples may be obtained from environmental ormanufacturing sources, e.g., food, water, soil, slurries, debris,biofilms from containers of aqueous fluids, airborne particles oraerosols, and the like, which may include processed samples, such asobtained from passing samples over or through a filtering device, orfollowing centrifugation, or by adherence to a medium, matrix, orsupport. Samples include “biological samples” which include any tissueor material derived from a living or dead mammal or organism, including,e.g., respiratory tissue or exudates such as bronchoscopy,bronchoalveolar lavage, lung biopsy, sputum, milk, peripheral blood,plasma, serum, lymph node, gastrointestinal tissue, feces, urine, orother body fluids or materials. A sample may be treated to physically ormechanically disrupt tissue aggregates or cells, thus releasingintracellular components, including target nucleic acids, into asolution which may contain other components, such as enzymes, buffers,salts, detergents and the like.

“Nucleic acid” refers to a multimeric compound comprising two or morecovalently bonded nucleosides or nucleoside analogs having nitrogenousheterocyclic bases, or base analogs, where the nucleosides are linkedtogether by phosphodiester bonds or other linkages to form apolynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNApolymers or oligonucleotides, and analogs thereof. A nucleic acid“backbone” may be made up of a variety of linkages, including one ormore of sugar-phosphodiester linkages, peptide-nucleic acid bonds (in“peptide nucleic acids” or PNAs, see PCT No. WO 95/32305),phosphorothioate linkages, methylphosphonate linkages, or combinationsthereof. Sugar moieties of the nucleic acid may be either ribose ordeoxyribose, or similar compounds having known substitutions, e.g., 2′methoxy substitutions and 2′ halide substitutions (e.g., 2′-F).Nitrogenous bases may be conventional bases (A, G, C, T, U), analogsthereof (e.g., inosine, 5-methylisocytosine, isoguanine; TheBiochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11^(th) ed.,1992, Abraham et al., 2007, BioTechniques 43: 617-24), which includederivatives of purine or pyrimidine bases (e.g., N⁴-methyldeoxygaunosine, deaza- or aza-purines, deaza- or aza-pyrimidines,pyrimidine bases having substituent groups at the 5 or 6 position,purine bases having an altered or replacement substituent at the 2, 6and/or 8 position, such as 2-amino-6-methylaminopurine,O⁶-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O⁴-alkyl-pyrimidines, andpyrazolo-compounds, such as unsubstituted or 3-substitutedpyrazolo[3,4-d]pyrimidine; U.S. Pat. Nos. 5,378,825, 6,949,367 and PCTNo. WO 93/13121). Nucleic acids may include “abasic” residues in whichthe backbone does not include a nitrogenous base for one or moreresidues (U.S. Pat. No. 5,585,481). A nucleic acid may comprise onlyconventional sugars, bases, and linkages as found in RNA and DNA, or mayinclude conventional components and substitutions (e.g., conventionalbases linked by a 2′ methoxy backbone, or a nucleic acid including amixture of conventional bases and one or more base analogs). Nucleicacids may include “locked nucleic acids” (LNA), in which one or morenucleotide monomers have a bicyclic furanose unit locked in an RNAmimicking sugar conformation, which enhances hybridization affinitytoward complementary sequences in single-stranded RNA (ssRNA),single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA) (Vester etal., 2004, Biochemistry 43(42):13233-41). Nucleic acids may includemodified bases to alter the function or behavior of the nucleic acid,e.g., addition of a 3′-terminal dideoxynucleotide to block additionalnucleotides from being added to the nucleic acid. Synthetic methods formaking nucleic acids in vitro are well known in the art although nucleicacids may be purified from natural sources using routine techniques.

The term “polynucleotide” as used herein denotes a nucleic acid chain.Throughout this application, nucleic acids are designated by the5′-terminus to the 3′-terminus. Standard nucleic acids, e.g., DNA andRNA, are typically synthesized “3′-to-5′,” i.e., by the addition ofnucleotides to the 5′-terminus of a growing nucleic acid.

A “nucleotide” as used herein is a subunit of a nucleic acid consistingof a phosphate group, a 5-carbon sugar and a nitrogenous base. The5-carbon sugar found in RNA is ribose. In DNA, the 5-carbon sugar is2′-deoxyribose. The term also includes analogs of such subunits, such asa methoxy group at the 2′ position of the ribose (2′-O-Me, or 2′methoxy). As used herein, methoxy oligonucleotides containing “T”residues have a methoxy group at the 2′ position of the ribose moiety,and a uracil at the base position of the nucleotide.

A “non-nucleotide unit” as used herein is a unit that does notsignificantly participate in hybridization of a polymer. Such units mustnot, for example, participate in any significant hydrogen bonding with anucleotide, and would exclude units having as a component one of thefive nucleotide bases or analogs thereof.

A “target nucleic acid” as used herein is a nucleic acid comprising a“target sequence” to be amplified. Target nucleic acids may be DNA orRNA and may be either single-stranded or double-stranded. The targetnucleic acid may include other sequences besides the target sequencethat may not be amplified. Typical target nucleic acids include virusgenomes, bacterial genomes, fungal genomes, plant genomes, animalgenomes, rRNA, tRNA, or mRNA from viruses, bacteria or eukaryotic cells,mitochondrial DNA, or chromosomal DNA. In the instant disclosure, targetnucleic acids are 16S rRNA or genes encoding 16S rRNA from one or moreof C. jejuni, C. lari, or C. coli.

By “isolated” it is meant that a target nucleic acid is taken from itsnatural milieu, but the term does not connote any degree ofpurification.

The term “target sequence” as used herein refers to the particularnucleotide sequence of the target nucleic acid that is to be amplified.Where the target nucleic acid is originally single-stranded, the term“target sequence” will also refer to the sequence complementary to thetarget sequence as present in the target nucleic acid. Where the targetnucleic acid is originally double-stranded, the term “target sequence”refers to both the sense (+) and antisense (−) strands. In choosing atarget sequence, the skilled artisan will understand that a sequenceshould be chosen so as to distinguish between unrelated or closelyrelated target nucleic acids.

The term “target a sequence,” “target(s) a target nucleic acid” or“targets a sequence” as used herein in reference to a region ofCampylobacter nucleic acid refers to a process whereby anoligonucleotide stably hybridizes to the target sequence in a mannerthat allows for amplification and/or detection as described herein. Inone embodiment, the oligonucleotide is complementary with the targetedCampylobacter nucleic acid sequence and contains no mismatches. Inanother embodiment, the oligonucleotide is complementary but contains 1;or 2; or 3; or 4; or 5 mismatches with the targeted Campylobacternucleic acid sequence. Preferably, the oligonucleotide that stablyhybridizes to the Campylobacter nucleic acid sequence includes at least10 to as many as 50 nucleotides complementary to the target sequence. Itis understood that at least 10 and as many as 50 is an inclusive rangesuch that 10, 50 and each whole number there between are included. Theterm “configured to target a sequence” as used herein means that thetarget hybridizing region of an amplification oligonucleotide isdesigned to have a polynucleotide sequence that could target a sequenceof the referenced Campylobacter region. Such an amplificationoligonucleotide is not limited to targeting that sequence only, but israther useful as a composition, in a kit or in a method for targeting aC. jejuni target nucleic acid and a C. coli, a C. lari or a C. coli anda C. lari target nucleic acid, as is described herein. The term“configured to” denotes an actual arrangement of the polynucleotidesequence configuration of the amplification oligonucleotide targethybridizing sequence.

The term “fragment” as used herein in reference to the Campylobactertargeted nucleic acid sequence refers to a piece of contiguous nucleicacid. In certain embodiments, the fragment includes contiguousnucleotides from a Campylobacter species' target nucleic acid, whereinthe number of contiguous nucleotides in the fragment are less than thatfor the entire 16S rRNA or its encoding gene.

The term “region” as used herein refers to a portion of a nucleic acidwherein said portion is smaller than the entire nucleic acid. Forexample, when the nucleic acid in reference is an oligonucleotidepromoter provider, the term “region” may be used refer to the smallerpromoter portion of the entire oligonucleotide. Similarly, and also asexample only, when the nucleic acid is a target nucleic acid, the term“region” may be used to refer to a smaller area of the nucleic acid.

The interchangeable terms “oligomer,” “oligo” and “oligonucleotide”refer to a nucleic acid having generally less than 1,000 nucleotide (nt)residues, including polymers in a range having a lower limit of about 5nt residues and an upper limit of about 500 to 900 nt residues. In someembodiments, oligonucleotides are in a size range having a lower limitof about 12 to 15 nt and an upper limit of about 50 to 600 nt, and otherembodiments are in a range having a lower limit of about 15 to 20 nt andan upper limit of about 22 to 100 nt. Oligonucleotides may be purifiedfrom naturally occurring sources, but or may be synthesized using any ofa variety of well known enzymatic or chemical methods. The termoligonucleotide does not denote any particular function to the reagent;rather, it is used generically to cover all such reagents describedherein. An oligonucleotide may serve various different functions. Forexample, it may function as a primer if it is specific for and capableof hybridizing to a complementary strand and can further be extended inthe presence of a nucleic acid polymerase, it may provide a promoter ifit contains a sequence recognized by an RNA polymerase and allows fortranscription (e.g., a T7 Provider), and it may function to preventhybridization or impede primer extension if appropriately situatedand/or modified.

As used herein, an oligonucleotide having a nucleic acid sequence“comprising” or “consisting of” or “consisting essentially of” asequence selected from a group of specific sequences means that theoligonucleotide, as a basic and novel characteristic, is capable ofstably hybridizing to a nucleic acid having the exact complement of oneof the listed nucleic acid sequences of the group under stringenthybridization conditions. An exact complement includes the correspondingDNA or RNA sequence.

As used herein, a nucleic acid “corresponds” to a specified nucleic acidif the nucleic acid is 100% identical or complementary to the specifiednucleic acid. As used herein, a nucleic acid “substantiallycorresponding to” a specified nucleic acid sequence means that thereferred to oligonucleotide is sufficiently similar to the referencenucleic acid sequence such that the oligonucleotide has similarhybridization properties to the reference nucleic acid sequence in thatit would hybridize with the same target nucleic acid sequence understringent hybridization conditions. Substantially corresponding nucleicacids vary by at least one nucleotide from the specified nucleic acid.This variation may be stated in terms of a percentage of identical orcomplementary between the nucleic acid and the specified nucleic acid.Thus, nucleic acid substantially corresponds to a reference nucleic acidsequence if these percentages of base identity or complementarity arefrom less than 100% to about 80%. In preferred embodiments, thepercentage is from less than 100% to about 85%. In more preferredembodiments, this percentage can be from less than 100% to about 90%; inother preferred embodiments, this percentage is from less than 100% toabout 95%. One skilled in the art will understand that the recitedranges include all whole and rational numbers of the range (e.g., 92% or98.377%).

A “helper oligonucleotide” or “helper” refers to an oligonucleotidedesigned to bind to a target nucleic acid and impose a differentsecondary and/or tertiary structure on the target to increase the rateand extent of hybridization of a detection probe or otheroligonucleotide with the targeted nucleic acid, as described, forexample, in U.S. Pat. No. 5,030,557. Helpers may also be used to assistwith the hybridization to target nucleic acid sequences and function ofprimer, target capture and other oligonucleotides.

As used herein, a “blocking moiety” is a substance used to “block” the3′-terminus of an oligonucleotide or other nucleic acid so that itcannot be efficiently extended by a nucleic acid polymerase.

An “amplification oligomer” is an oligomer, at least the 3′-end of whichis complementary to a target nucleic acid (“target hybridizingsequence”), and which hybridizes to a target nucleic acid, or itscomplement, and participates in a nucleic acid amplification reaction.An example of an amplification oligomer is a “primer” that hybridizes toa target nucleic acid and contains a 3′ OH end that is extended by apolymerase in an amplification process. Another example of anamplification oligomer is a “promoter-based amplification oligomer,”which comprises a target hybridizing sequence, and a promoter sequencefor initiating transcription by an appropriate polymerase.Promoter-based amplification oligomers may or may not be extended by apolymerase in a primer-based extension depending upon whether or not the3′ end of the target hybridizing sequence is modified to preventprimer-based extension (e.g., a 3′ blocked end). A promoter-basedamplification oligonucleotide comprising a target hybridizing regionthat is not modified to prevent primer-based extension is referred to asa “promoter-primer.” A promoter-based amplification oligonucleotidecomprising a target hybridizing region that is modified to preventprimer-based extension is referred to as a “promoter-provider.” Sizeranges for amplification oligonucleotides include those comprisingtarget hybridizing regions that are about 10 to about 70 nt long.Included in this range are all whole numbers of the range, as isunderstood by a skilled artisan (e.g., 10, 11, 12, 13 . . . 67, 68, 69and 70). An amplification oligomer may optionally include modifiednucleotides or analogs that are not complementary to target nucleic acidin a strict A:T/U, G:C sense. Such modified nucleotides or analogs areherein considered mismatched to their corresponding target sequence.

Oligomers not intended for primer-based extension by a nucleic acidpolymerase may include a blocker group that replaces the 3′ OH toprevent the enzyme-mediated extension of the oligomer in anamplification reaction. For example, blocked amplification oligomersand/or detection probes present during amplification may not havefunctional 3′OH and instead include one or more blocking groups locatedat or near the 3′ end. In some embodiments a blocking group near the 3′end and may be within five residues of the 3′ end and is sufficientlylarge to limit binding of a polymerase to the oligomer. In otherembodiments a blocking group is covalently attached to the 3′ terminus.Many different chemical groups may be used to block the 3′ end, e.g.,alkyl groups, non-nucleotide linkers, alkane-diol dideoxynucleotideresidues, and cordycepin.

As used herein, a “promoter” is a specific nucleic acid sequence that isrecognized by a DNA-dependent RNA polymerase (“transcriptase”) as asignal to bind to the nucleic acid and begin the transcription of RNA ata specific site.

As used herein, a “promoter-provider” or “provider” refers to anoligonucleotide comprising first and second regions, and which ismodified to prevent the initiation of DNA synthesis from its3′-terminus. The “first region” of a promoter-provider oligonucleotidecomprises a base sequence which hybridizes to a DNA template, where thehybridizing sequence is situated 3′, but not necessarily adjacent to, apromoter region. The target-hybridizing portion of a promoteroligonucleotide is typically at least 10 nucleotides in length, and mayextend up to 50 or more nucleotides in length. The “second region”comprises a promoter sequence for an RNA polymerase. A promoter-provideroligonucleotide is configured so that it is incapable of being extendedby an RNA- or DNA-dependent DNA polymerase, (e.g., reversetranscriptase), preferably by comprising a blocking moiety at its3′-terminus as described above. This modification differentiatespromoter providers from promoter primers. Preferably, the promoterportion of a promoter primer or provider is a promoter for aDNA-dependent RNA polymerase from E. coli and bacteriophages T7, T3, andSP6, though other promoters or modified version thereof can be used aswell.

As used herein, a “terminating oligonucleotide” or “blockeroligonucleotide” is an oligonucleotide comprising a base sequence thatis complementary to a region of the target nucleic acid in the vicinityof the 5′-end of the target sequence, so as to “terminate” primerextension of a nascent nucleic acid that includes a primingoligonucleotide, thereby providing a defined 3′-end for the nascentnucleic acid strand.

“Amplification” refers to any known procedure for obtaining multiplecopies of a target nucleic acid sequence or its complement or fragmentsthereof. The multiple copies may be referred to as amplicons oramplification products. Amplification of “fragments” refers toproduction of an amplified nucleic acid that contains less than thecomplete target nucleic acid or its complement, e.g., produced by usingan amplification oligonucleotide that hybridizes to, and initiatespolymerization from, an internal position of the target nucleic acid.Known amplification methods include, for example, replicase-mediatedamplification, polymerase chain reaction (PCR), ligase chain reaction(LCR), strand-displacement amplification (SDA), andtranscription-mediated or transcription-associated amplification.Replicase-mediated amplification uses self-replicating RNA molecules,and a replicase such as QB-replicase (e.g., U.S. Pat. No. 4,786,600).PCR amplification uses a DNA polymerase, pairs of primers, and thermalcycling to synthesize multiple copies of two complementary strands ofdsDNA or from a cDNA (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, and4,800,159). LCR amplification uses four or more differentoligonucleotides to amplify a target and its complementary strand byusing multiple cycles of hybridization, ligation, and denaturation(e.g., U.S. Pat. Nos. 5,427,930 and 5,516,663). SDA uses a primer thatcontains a recognition site for a restriction endonuclease and anendonuclease that nicks one strand of a hemimodified DNA duplex thatincludes the target sequence, whereby amplification occurs in a seriesof primer extension and strand displacement steps (e.g., U.S. Pat. Nos.5,422,252; 5,547,861; and 5,648,211).

“Transcription associated amplification” or “transcription mediatedamplification” (TMA) refer to nucleic acid amplification that uses anRNA polymerase to produce multiple RNA transcripts from a nucleic acidtemplate. These methods generally employ an RNA polymerase, a DNApolymerase, deoxyribonucleoside triphosphates, ribonucleosidetriphosphates, and a template complementary oligonucleotide thatincludes a promoter sequence, and optionally may include one or moreother oligonucleotides. TMA methods are embodiments of amplificationmethods used for amplifying and detecting C. jejuni, C. lari, or C. colitarget sequences as described herein. Variations of transcriptionassociated amplification are well known in the art as previouslydisclosed in detail (e.g., U.S. Pat. Nos. 4,868,105; 5,124,246;5,130,238; 5,399,491; 5,437,990; 5,554,516; and 7,374,885; and PCT Pub.Nos. WO 88/01302; WO 88/10315 and WO 95/03430). The person of ordinaryskill in the art will appreciate that the disclosed compositions may beused in amplification methods based on extension of oligomer sequencesby a polymerase.

As used herein, the term “real-time TMA” refers to single-primertranscription-mediated amplification (“TMA”) of target nucleic acid thatis monitored by real-time detection means.

The term “amplicon” or the term “amplification product” as used hereinrefers to the nucleic acid molecule generated during an amplificationprocedure that is complementary or homologous to a sequence containedwithin the target sequence. These terms can be used to refer to a singlestrand amplification product, a double strand amplification product orone of the strands of a double strand amplification product.

“Probe,” “detection probe” or “detection oligonucleotide” are termsreferring to a nucleic acid oligomer that hybridizes specifically to atarget sequence in a nucleic acid, or in an amplified nucleic acid,under conditions that promote hybridization to allow detection of thetarget sequence or amplified nucleic acid. Detection may either bedirect (e.g., a probe hybridized directly to its target sequence) orindirect (e.g., a probe linked to its target via an intermediatemolecular structure). Probes may be DNA, RNA, analogs thereof orcombinations thereof and they may be labeled or unlabeled. A probe's“target sequence” generally refers to a smaller nucleic acid sequencewithin a larger nucleic acid sequence that hybridizes specifically to atleast a portion of a probe oligomer by standard base pairing. A probemay comprise target-specific sequences and other sequences thatcontribute to the three-dimensional conformation of the probe (e.g.,U.S. Pat. Nos. 5,118,801; 5,312,728; 6,849,412; 6,835,542; 6,534,274;and 6,361,945; and US Pub. No. 20060068417).

By “stable” or “stable for detection” is meant that the temperature of areaction mixture is at least 2° C. below the melting temperature of anucleic acid duplex.

As used herein, a “label” refers to a moiety or compound joined directlyor indirectly to a probe that is detected or leads to a detectablesignal. Direct labeling can occur through bonds or interactions thatlink the label to the probe, including covalent bonds or non-covalentinteractions, e.g. hydrogen bonds, hydrophobic and ionic interactions,or formation of chelates or coordination complexes. Indirect labelingcan occur through use of a bridging moiety or “linker” such as a bindingpair member, an antibody or additional oligomer, which is eitherdirectly or indirectly labeled, and which may amplify the detectablesignal. Labels include any detectable moiety, such as a radionuclide,ligand (e.g., biotin, avidin), enzyme or enzyme substrate, reactivegroup, or chromophore (e.g., dye, particle, or bead that impartsdetectable color), luminescent compound (e.g., bioluminescent,phosphorescent, or chemiluminescent labels), or fluorophore. Labels maybe detectable in a homogeneous assay in which bound labeled probe in amixture exhibits a detectable change different from that of an unboundlabeled probe, e.g., instability or differential degradation properties.A “homogeneous detectable label” can be detected without physicallyremoving bound from unbound forms of the label or labeled probe (e.g.,U.S. Pat. Nos. 5,283,174, 5,656,207, and 5,658,737). Labels includechemiluminescent compounds, e.g., acridinium ester (“AE”) compounds thatinclude standard AE and derivatives (e.g., U.S. Pat. Nos. 5,656,207,5,658,737, and 5,639,604). Synthesis and methods of attaching labels tonucleic acids and detecting labels are well known (e.g., Sambrook etal., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989), Chapter 10; U.S. Pat.Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, and 4,581,333). Morethan one label, and more than one type of label, may be present on aparticular probe, or detection may use a mixture of probes in which eachprobe is labeled with a compound that produces a detectable signal(e.g., U.S. Pat. Nos. 6,180,340 and 6,350,579).

As used herein, structures referred to as “molecular torches” aredesigned to include distinct regions of self-complementarity (“theclosing domain”) which are connected by a joining region (“the targetbinding domain”) and which hybridize to one another under predeterminedhybridization assay conditions. All or part of the nucleotide sequencescomprising target closing domains may also function as target bindingdomains. Thus, target closing sequences can include, target bindingsequences, non-target binding sequences, and combinations thereof.

As used herein, a “capture oligonucleotide” or “capture probe” refers toa nucleic acid oligomer that specifically hybridizes to a targetsequence in a target nucleic acid by standard base pairing and joins toa binding partner on an immobilized probe to capture the target nucleicacid to a support. One example of a capture oligomer includes anoligonucleotide comprising two binding regions: a target hybridizingsequence and an immobilized probe-binding region. A variation of thisexample, the two regions may be present on two different oligomersjoined together by one or more linkers. Another embodiment of a captureoligomer the target hybridizing sequence is a sequence that includesrandom or non-random poly-GU, poly-GT, or poly U sequences to bindnon-specifically to a target nucleic acid and link it to an immobilizedprobe on a support. (PCT Pub No. WO 2008/016988). The immobilized probebinding region can be a nucleic acid sequence, referred to as a tail.Tails include a substantially homopolymeric tail of about 10 to 40nucleotides (e.g., A₁₀ to A₄₀), or of about 14 to 33 nt (e.g., T₃A₁₄ toT₃A₃₀), that bind to a complementary immobilized sequence attached tothe support particle or support matrix. Another example of a of acapture oligomer comprises two regions, a target hybridizing sequenceand a binding pair member that is not a nucleic acid sequence.

As used herein, an “immobilized oligonucleotide”, “immobilized probe” or“immobilized nucleic acid” refers to a nucleic acid binding partner thatjoins a capture oligomer to a support, directly or indirectly. Animmobilized probe joined to a support facilitates separation of acapture probe bound target from unbound material in a sample. Oneembodiment of an immobilized probe is an oligomer joined to a supportthat facilitates separation of bound target sequence from unboundmaterial in a sample. Supports may include known materials, such asmatrices and particles free in solution, which may be made ofnitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene,silane, polypropylene, metal, or other compositions, of which oneembodiment is magnetically attractable particles. Supports may bemonodisperse magnetic spheres (e.g., uniform size ±5%), to which animmobilized probe is joined directly (via covalent linkage, chelation,or ionic interaction), or indirectly (via one or more linkers), wherethe linkage or interaction between the probe and support is stableduring hybridization conditions.

By “complementary” is meant that the nucleotide sequences of similarregions of two single-stranded nucleic acids, or to different regions ofthe same single-stranded nucleic acid have a nucleotide base compositionthat allow the single-stranded regions to hybridize together in a stabledouble-stranded hydrogen-bonded region under stringent hybridization oramplification conditions. Sequences that hybridize to each other may becompletely complementary or partially complementary to the intendedtarget sequence by standard nucleic acid base pairing (e.g. G:C, A:T orA:U pairing). By “sufficiently complementary” is meant a contiguoussequence that is capable of hybridizing to another sequence by hydrogenbonding between a series of complementary bases, which may becomplementary at each position in the sequence by standard base pairingor may contain one or more residues that are not complementary bystandard A:T/U and G:C pairing, or are modified nucleotides such asabasic residues, modified nucleotides or nucleotide analogs.Sufficiently complementary contiguous sequences typically are at least80%, or at least 90%, complementary to a sequence to which an oligomeris intended to specifically hybridize (a %-complementarity rangeincludes all whole and rational numbers of the range). Sequences thatare “sufficiently complementary” allow stable hybridization of a nucleicacid oligomer with its target sequence under appropriate hybridizationconditions, even if the sequences are not completely complementary. Whena contiguous sequence of nucleotides of one single-stranded region isable to form a series of “canonical” hydrogen-bonded base pairs with ananalogous sequence of nucleotides of the other single-stranded region,such that A is paired with U or T and C is paired with G, thenucleotides sequences arc “completely” complementary.

By “preferentially hybridize” is meant that under stringenthybridization assay conditions, an oligonucleotide hybridizes to itstarget sequences, or replicates thereof, to form stable oligonucleotide:target sequence hybrid, while at the same time formation of stableoligonucleotide: non-target sequence hybrid is minimized. For example, aprobe oligonucleotide preferentially hybridizes to a target sequence orreplicate thereof to a sufficiently greater extent than to a non-targetsequence, to enable one having ordinary skill in the art to accuratelydetect the RNA replicates or complementary DNA (cDNA) of the targetsequence formed during the amplification. Appropriate hybridizationconditions are well known in the art for probe, amplification, targetcapture, blocker and other oligonucleotides, may be predicted based onsequence composition, or can be determined by using routine testingmethods (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual,2^(nd) ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989) at §§ 1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57,particularly §§ 9.50-9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57).

By “nucleic acid hybrid” or “hybrid” or “duplex” is meant a nucleic acidstructure containing a double-stranded, hydrogen-bonded region whereineach strand is complementary to the other, and wherein the region issufficiently stable under stringent hybridization conditions to bedetected by means including, but not limited to, chemiluminescent orfluorescent light detection, autoradiography, or gel electrophoresis.Such hybrids may comprise RNA:RNA, RNA:DNA, or DNA:DNA duplex molecules.

“Sample preparation” refers to any steps or methods that treat a samplefor subsequent amplification and/or detection of C. jejuni, C. lari, orC. coli nucleic acids present in the sample. Samples may be complexmixtures of components of which the target nucleic acid is a minoritycomponent. Sample preparation may include any known method ofconcentrating components, such as microbes or nucleic acids, from alarger sample volume, such as by filtration of airborne or waterborneparticles from a larger volume sample or by isolation of microbes from asample by using standard microbiology methods. Sample preparation mayinclude physical disruption and/or chemical lysis of cellular componentsto release intracellular components into a substantially aqueous ororganic phase and removal of debris, such as by using filtration,centrifugation or adsorption. Sample preparation may include use ofnucleic acid oligonucleotide that selectively or non-specificallycapture a target nucleic acid and separate it from other samplecomponents (e.g., as described in U.S. Pat. No. 6,110,678 and PCT Pub.No. WO 2008/016988).

“Separating” or “purifying” means that one or more components of asample are removed or separated from other sample components. Samplecomponents include target nucleic acids usually in a generally aqueoussolution phase, which may also include cellular fragments, proteins,carbohydrates, lipids, and other nucleic acids. Separating or purifyingremoves at least 70%, or at least 80%, or at least 95% of the targetnucleic acid from other sample components. Ranges of %-purity includeall whole and rational numbers of the range.

As used herein, a “DNA-dependent DNA polymerase” is an enzyme thatsynthesizes a complementary DNA copy from a DNA template. Examples areDNA polymerase I from E. coli, bacteriophage T7 DNA polymerase, or DNApolymerases from bacteriophages T4, Phi-29, M2, or T5. DNA-dependent DNApolymerases may be the naturally occurring enzymes isolated frombacteria or bacteriophages or expressed recombinantly, or may bemodified or “evolved” forms which have been engineered to possesscertain desirable characteristics, e.g., thermostability, or the abilityto recognize or synthesize a DNA strand from various modified templates.All known DNA-dependent DNA polymerases require a complementary primerto initiate synthesis. It is known that under suitable conditions aDNA-dependent DNA polymerase may synthesize a complementary DNA copyfrom an RNA template. RNA-dependent DNA polymerases typically also haveDNA-dependent DNA polymerase activity.

As used herein, a “DNA-dependent RNA polymerase” or “transcriptase” isan enzyme that synthesizes multiple RNA copies from a double-stranded orpartially double-stranded DNA molecule having a promoter sequence thatis usually double-stranded. The RNA molecules (“transcripts”) aresynthesized in the 5′-to-3′ direction beginning at a specific positionjust downstream of the promoter. Examples of transcriptases are theDNA-dependent RNA polymerase from E. coli and bacteriophages T7, T3, andSP6.

As used herein, an “RNA-dependent DNA polymerase” or “reversetranscriptase” (“RT”) is an enzyme that synthesizes a complementary DNAcopy from an RNA template. All known reverse transcriptases also havethe ability to make a complementary DNA copy from a DNA template; thus,they are both RNA- and DNA-dependent DNA polymerases. RTs may also havean RNAse H activity. A primer is required to initiate synthesis withboth RNA and DNA templates.

As used herein, a “selective RNAse” is an enzyme that degrades the RNAportion of an RNA:DNA duplex but not single-stranded RNA,double-stranded RNA or DNA. An exemplary selective RNAse is RNAse H.Enzymes possessing the same or similar activity as RNAse H may also beused. Selective RNAses may be endonucleases or exonucleases. Mostreverse transcriptase enzymes contain an RNAse H activity in addition totheir polymerase activities. However, other sources of the RNAse H areavailable without an associated polymerase activity. The degradation mayresult in separation of RNA from a RNA:DNA complex. Alternatively, aselective RNAse may simply cut the RNA at various locations such thatportions of the RNA melt off or permit enzymes to unwind portions of theRNA. Other enzymes that selectively degrade RNA target sequences or RNAproducts of the present invention will be readily apparent to those ofordinary skill in the art.

The term “specificity,” in the context of an amplification system, isused herein to refer to the characteristic of an amplification systemwhich describes its ability to distinguish between target and non-targetsequences dependent on sequence and assay conditions. In terms ofnucleic acid amplification, specificity generally refers to the ratio ofthe number of specific amplicons produced to the number of side-products(e.g., the signal-to-noise ratio).

The term “sensitivity” is used herein to refer to the precision withwhich a nucleic acid amplification reaction can be detected orquantitated. The sensitivity of an amplification reaction is generally ameasure of the smallest copy number of the target nucleic acid that canbe reliably detected in the amplification system, and will depend, forexample, on the detection assay being employed, and the specificity ofthe amplification reaction, e.g., the ratio of specific amplicons toside-products.

As used herein, a “colony forming unit” (“CFU”) is used as a measure ofviable microorganisms in a sample. A CFU is an individual viable cellcapable of forming on a solid medium a visible colony whose individualcells are derived by cell division from one parental cell. One CFUcorresponds to 1000 copies of rRNA.

As used herein, the term “TTime” is the threshold time or time ofemergence of signal in a real-time plot of the assay data. TTime valuesestimate the time at which a particular threshold indicating ampliconproduction is passed in a real-time amplification reaction. TTime and analgorithm for calculating and using TTime values are described in U.S.Pub. No. 2006/0276972, paragraphs [0517] through [0538]. A curve fittingprocedure is applied to normalized and background-adjusted data. Thecurve fit is performed for only a portion of the data between apredetermined low bound and high bound. The goal, after finding thecurve that fits the data, is to estimate the time corresponding to thepoint at which the curve or a projection thereof intersects a predefinedthreshold value. In one embodiment, the threshold for normalized data is0.11. The high and low bounds are determined empirically as that rangeover which curves fit to a variety of control data sets exhibit theleast variability in the time associated with the given threshold value.For example, in one embodiment, the low bound is 0.04 and the high boundis 0.36. The curve is fit for data extending from the first data pointbelow the low bound through the first data point past the high bound.Next, there is made a determination whether the slope of the fit isstatistically significant. For example, if the p value of the firstorder coefficient is less than 0.05, the fit is considered significant,and processing continues. If not, processing stops. Alternatively, thevalidity of the data can be determined by the R² value. The slope m andintercept b of the linear curve y=mx+b are determined for the fittedcurve. With that information, TTime can be determined as follows:

TTime=(Threshold−b)/m

As used herein, the term “relative fluorescence unit” (“RFU”) is anarbitrary unit of measurement of fluorescence intensity. RFU varies withthe characteristics of the detection means used for the measurement.

Transcription Mediated Amplification and Real-Time TranscriptionMediated Amplification.

Amplification methods that use TMA amplification include the followingsteps. Briefly, a single stranded target nucleic acid containing thetarget sequence to be amplified is provided. Those skilled in the artwill appreciate that conventional melting of double stranded nucleicacid may be used to provide single-stranded target nucleic acids. Afirst amplification oligomer is brought in contact with that targetnucleic acid by hybridizing to the target sequence. The firstamplification oligomer may be a primer or a promoter primer. A suitablenucleic acid polymerase then generates a nucleic acid strandamplification product that is complementary to the target nucleic acidtarget sequence. In the instances where the target nucleic acid is anRNA, the RNA is typically degraded leaving just the newly generatedamplification product, which is available for hybridization by a secondamplification oligomer. Using a primer as the first amplificationoligomer, then the second amplification oligomer is a promoter primer orpromoter provider. A suitable nucleic acid polymerase uses the newlygenerated amplification product to which the promoter-based oligomer ishybridized as a primer to make a complementary strand of theunhybridized promoter sequence. If the second amplification oligomer isa promoter primer, then a complementary copy of the amplificationproduct hybridized by the second amplification oligomer is alsogenerated. The now double stranded promoter sequence of thepromoter-based amplification is used by a suitable RNA polymerase toinitiate transcription and make RNA transcript amplification products.The first amplification oligomer primer can then hybridize thetranscribed amplification products and the steps can repeat. Or, thetarget nucleic acid is RNA and the first amplification oligomer is apromoter-based amplification oligomer. Here, the promoter basedamplification oligomer is a promoter primer. A suitable polymerase makesa first amplification product that is complementary to the RNA targetsequence. The RNA target nucleic acid is degraded and a secondamplification oligomer is hybridized to the amplification product. Asuitable polymerase makes a complement strand, thereby generating adouble stranded promoter sequence. Transcription is initiated and RNA istranscribed. The transcribed RNA is complementary to the original targetnucleic acid, thus the second amplification oligomer hybridizes againand makes the transcribed RNA double stranded. The RNA is degraded andthe remaining DNA strand is hybridized by the first amplificationoligomer. The amplification steps can repeat. When the target nucleicacid is DNA the first amplification oligomer is a promoter primer andthe second amplification is a primer. Amplification generally proceedsas described above, and as is described in the art. See e.g., U.S. Pat.Nos. 4,868,105; 5,124,246; 5,130,238; 5,399,491; 5,437,990; 5,554,516;and 7,374,885; and PCT Pub. Nos. WO 88/01302; WO 88/10315 and WO95/03430 describing TMA and other variations of transcription-associatedamplification. The amplified products may be detected in real-timeduring amplification, or at the end of the amplification reaction.Detection may be performed by a number of methods. Probe-based detectionmethods use an oligonucleotide probe comprising a target hybridizingsequence that binds specifically to a target sequence contained in theamplification products. Detection of a signal resulting from the boundprobes indicates the presence of the target nucleic acid in the sample.

Nucleic Acid Detection.

Detection of the nucleic acids may be accomplished by a variety ofmethods. Detection methods may use nucleic acid probes comprising atarget hybridizing sequence that is complementary to a portion of theamplified product and detecting the presence of the probe:productcomplex, or by using a complex of probes that may amplify the detectablesignal associated with the amplified products (e.g., U.S. Pat. Nos.5,424,413; 5,451,503; and 5,849,481). Directly or indirectly labeledprobes that specifically associate with the amplified product provide adetectable signal that indicates the presence of the target nucleic acidin the sample. For example, if the target nucleic acid is C. jejuni, C.lari, or C. coli 16S rRNA, the amplified product will contain a sequencein or complementary to a C. jejuni, C. lari, or C. coli target sequence.A probe is configured to bind directly or indirectly to a portion of theamplification product to indicate the presence of C. jejuni, C. lari, orC. coli in the tested sample.

Embodiments of probes that hybridize to the amplified sequences includehairpin oligonucleotides such as Molecular Torches and linearoligonucleotides that substantially do not form conformations held byintramolecular bonds. Preferably, said probes may include labels. Linearprobe embodiments may include a chemiluminescent compound as the label,e.g. a chemiluminescent AE compound attached to the probe sequence via alinker (substantially as described in U.S. Pat. Nos. 5,585,481 and5,639,604, particularly at column 10, line 6 to column 11, line 3, andin Example 8 therein). Examples of labeling positions are a centralregion of the probe oligomer and near a region of A:T base pairing, at a3′ or 5′ terminus of the oligomer, and at or near a mismatch site with aknown sequence that is not the desired target sequence. Hairpin orlinear probes may be labeled with any of a variety of different types ofinteracting labels, where one interacting member is usually attached tothe 5′ end of the probe and the other interacting member is attached tothe 3′ end of the probe. Dye labeled probes, including dual labeledprobes, single labeled probes, AE labeled probes and the like, aregenerally known. dual labeled probes can be labeled at one end with afluorescent label (“F”) that absorbs light of a particular wavelength orrange and emits light another emission wavelength or range and at theother end with a quencher (“Q”) that dampens, partially or completely,signal emitted from the excited F when Q is in proximity with thefluorophore. Such a probe may be referred to as labeled with afluorescent/quencher (F/Q) pair. One embodiment of a hairpin probe is a“molecular torch” that detects an amplified product to indicate whethera target C. jejuni, C. lari, or C. coli sequence is present in thesample after the amplification step. A molecular torch probe comprises atarget binding domain and a closing domain, as is described above. Thesedomains allow the molecular torch to exist in open and closedconformations, depending on whether the torch is bound to a target. (Seealso, U.S. Pat. Nos. 6,849,412; 6,835,542; 6,534,274; and 6,361,945).Another hairpin probe embodiment is a “molecular beacon” which isgenerally described in Tyagi et al., 1998, Nature Biotechnol. 16:49-53,and in U.S. Pat. Nos. 5,118,801; and 5,312,728. Methods for using suchhairpin probes to detect the presence of a target sequence are wellknown in the art.

A method for detecting C. jejuni, C. lari, or C. coli sequences uses atranscription associated amplification and a molecular torch. Themolecular torch is added before or during amplification, allowingdetection to be carried out without the addition of other reagents. Forexample, a molecular torch may be designed so that the Tm of thehybridized target binding region and closing region complex is higherthan the amplification reaction temperature, thusly designed to preventthe probe from prematurely binding to amplified target sequences. Afteran interval of amplification, the mixture is heated to open the torchregions and allow the target binding regions to hybridize to a portionof the amplification products. The solution is then cooled to close anyprobes not bound to amplified products by allowing the probe targetbinding and closing regions to hybridize, which effectively closes thelabel/quencher pair. Detection is then performed to generate and detectsignals from only the probes that are hybridized to the amplified targetsequences. For example, the mixture containing the F/Q labeled hairpinprobe is irradiated with the appropriate excitation light and theemission signal is measured. In other embodiments, the hairpin detectionprobe is designed so that the amplified products hybridize to the targetbinding region of the probe during amplification, resulting in changingthe hairpin to its open conformation during amplification, and theamplification reaction mixture is irradiated at intervals to detect theemitted signal from the open probes in real time during amplification.

Sample Preparation.

Preparation of samples for amplification and detection of C. jejuni, C.lari, or C. coli sequences may include methods of separating and/orconcentrating organisms contained in a sample from other samplecomponents, e.g., filtration of particulate matter from air, water orother types of samples. Sample preparation may include routine methodsof disrupting cells or lysing bacteria to release intracellularcontents, including C. jejuni, C. lari, or C. coli 16S rRNA or geneticsequences encoding 16S rRNA. Sample preparation before amplification mayinclude an optional step of target capture to specifically ornon-specifically separate the target nucleic acids from other samplecomponents. Nonspecific target capture methods may involve selectiveprecipitation of nucleic acids from a substantially aqueous mixture,adherence of nucleic acids to a support that is washed to remove othersample components, other methods of physically separating nucleic acidsfrom a mixture that contains C. jejuni, C. lari, or C. coli nucleic acidand other sample components.

In one embodiment, C. jejuni, C. lari, or C. coli target nucleic acidsare selectively separated from other sample components by specificallyhybridizing the C. jejuni, C. lari, or C. coli target nucleic acid to acapture oligomer specific for C. jejuni, C. lari, and/or C. coli to forma target sequence:capture probe complex. The complex is separated fromsample components by binding the target:capture probe complex to animmobilized probe, and separating the target:capture probe:immobilizedprobe complex from the sample, as previously described (U.S. Pat. Nos.6,110,678; 6,280,952; and 6,534,273). Target capture may occur in asolution phase mixture that contains one or more captureoligonucleotides that hybridize specifically to target nucleic acidsunder hybridizing conditions, usually at a temperature higher than theTm of the tail sequence:immobilized probe sequence duplex. Thetarget:capture probe complex is captured by adjusting the hybridizationconditions so that the capture probe tail hybridizes to the immobilizedprobe, and the entire complex on the support is then separated fromother sample components. The support with the attached immobilizedprobe:capture probe:target sequence may be washed one or more times tofurther remove other sample components. Other embodiments link theimmobilized probe to a particulate support, such as a paramagnetic bead,so that particles with the attached target:capture probe:immobilizedprobe complex may be suspended in a washing solution and retrieved fromthe washing solution, by using magnetic attraction. To limit the numberof handling steps, the target nucleic acid may be amplified by simplymixing the target sequence in the complex on the support withamplification oligonucleotides and proceeding with amplification steps.

Preferred Campylobacter Oligonucleotides for the Amplification andDetection of 16S rRNA Sequences of C. jejuni, C. Lari, or C. coli.

As is described herein, preferred sites for selectively amplifying anddetecting Campylobacter jejuni, Campylobacter lari and Campylobactercoli nucleic acids are disclosed. The preferred sites are selected todiscriminate against amplification and/or detection of otherCampylobacter species and other bacteria (herein referred to as “nearneighbor organisms,” “challenge organisms” or “exclusionaries”). Thesepreferred sites are found within a region of the Campylobacter 16S rRNA.Particularly preferred oligonucleotides and oligonucleotide sets withthese regions have been identified for amplifying Campylobacter jejuni,Campylobacter lari and Campylobacter coli 16S rRNA with improvedsensitivity, selectivity and specificity.

Oligonucleotides were designed by first comparing 16S rRNA sequences (orgene sequences encoding 16S rRNA) of a number of Campylobacter species(e.g., C. jejuni, C. lari, C. coli, C. fetus, C. upsaliensis, C.hyointestinalis, C. curvus, C. hominis, C. insulaenigrae, C. lanienae,C. mucosalis, C. rectus, C. sputorum, and C. concisus) and a number ofother bacterial species within the order of Campylobacterales (e.g.,Arcobacter, Helicobacter, Flexispira, Geospirillum, Sulfurospirillum,Sulfuricurvum, and Hydrogenimonas). Oligonucleotides were synthesized invitro, and in some embodiment oligomers can be characterized bydetermining the Tm and hybridization characteristics of the C. jejuni,C. lari, or C. coli oligomers with complementary target sequences(synthetic or purified rRNA from bacteria) using standard laboratorymethods. Then, selected oligomer sequences were further tested against16S rRNA sequences. This test used different combinations ofamplification oligomers (selected from those shown in Table 1). Theamplification reactions included 16S rRNA targets from lysates orpurified from various Campylobacter species grown in culture. Theamplification reaction determined the efficiency of amplification of the16S rRNA target sequences using the various amplification oligocombinations. Amplification oligomers include those that may function asprimer, promoter primer, and promoter provider oligomers. The relativeefficiencies of different combinations of amplification oligomers weremonitored by detecting the amplified products of the amplificationreactions, generally by binding a labeled probe (Table 2) to theamplified products and detecting the relative amount of signal thatindicated the amount of amplified product made.

Some examples of primers useful with the current invention includeoligonucleotides that are from about 10 to about 70 nucleotides inlength and comprise a nucleotide sequence that is designed to hybridizeto either the “+” or the “−” strand of a region of SEQ ID NO:91, whereinthe region of SEQ ID NO:91 is from about nucleotide 62 to aboutnucleotide 226; is from about nucleotide 170 to about nucleotide 226; isfrom about nucleotide 170 to about nucleotide 205 and the primerincludes a sequence hybridizing to nucleotide 184 to nucleotide 194; isfrom about nucleotide 184 to about nucleotide 212 and the primerincludes a sequence that targets nucleotide 192 to nucleotide 205; isfrom about nucleotide 170 to about nucleotide 212; or is from aboutnucleotide 170 to about nucleotide 226 and the primer includes sequencehybridizing to nucleotide 184 to nucleotide 194, a sequence hybridizingto nucleotide 192 to nucleotide 205 or both. (See, SEQ ID NOS:77-83)Additions, deletions and mismatches may be incorporated into a primeroligo designed to hybridize to a region of SEQ ID NO:91. Representativeprimer oligo sequences are shown in Table 1 as SEQ ID NOS:1-17(Primers).

Some embodiments of the target-specific sequence of a promoterprimer/promoter provider oligomer are listed in Table 1, and thesetarget-specific sequences may be attached to the 3′ end of any knownpromoter sequence. The promoter sequences of a promoter primer/promoterprovider will be shown in lower case in Table 1. An example of apromoter sequence specific for the RNA polymerase of bacteriophage T7 isSEQ ID NO:75 (5′-aatttaatacgactcactatagggaga). Other promoter sequencesare also useful for promoter primer/promoter provider oligos. Someexamples of promoter-based amplification oligomer target hybridizingsequences include oligonucleotides that are from about 10 to about 50nucleotides in length and comprise a nucleotide sequence that isconfigured to hybridize to either the “+” or the “−” strand of a regionof SEQ ID NO:91. One region of SEQ ID NO:91 is from about nucleotide 14to about nucleotide 150. One region is from about nucleotide 14 to aboutnucleotide 41 of SEQ ID NO:91. One region is from about nucleotide 14 toabout nucleotide 41 of SEQ ID NO:91 and the target hybridizing sequencecontains a sequence identical or complementary to nucleotide 20 tonucleotide 37 of SEQ ID NO:91. One region of SEQ ID NO:91 is from aboutnucleotide 58 to about nucleotide 108. One region of SEQ ID NO:91 isfrom about nucleotide 108 to about nucleotide 150. One region of SEQ IDNO:91 is from about nucleotide 108 to about nucleotide 150 and thetarget hybridizing sequence contains a sequence identical orcomplementary to nucleotide 125 to nucleotide 135 of SEQ ID NO:91. Oneregion of SEQ ID NO:91 is from about nucleotide 58 to about nucleotide150. One region of SEQ ID NO:91 is from about nucleotide 112 to aboutnucleotide 138. One region of SEQ ID NO:91 is from about nucleotide 112to about nucleotide 138 and the target hybridizing sequence contains asequence identical or complementary to nucleotide 125 to nucleotide 135of SEQ ID NO:91. One region of SEQ ID NO:91 is from about nucleotide 112to about nucleotide 138 and the target hybridizing sequence contains asequence identical or complementary to nucleotide 115 to nucleotide 135of SEQ ID NO:91. One region of SEQ ID NO:91 is from about nucleotide 115to about nucleotide 150. One region of SEQ ID NO:91 is from aboutnucleotide 115 to about nucleotide 150 and the target hybridizingsequence contains a sequence identical or complementary to nucleotide125 to nucleotide 135 of SEQ ID NO:91. One region of SEQ ID NO:91 isfrom about nucleotide 125 to about nucleotide 150. One region of SEQ IDNO:91 is from about nucleotide 83 to about nucleotide 150. One region ofSEQ ID NO:91 is from about nucleotide 108 to about nucleotide 150 andthe target hybridizing sequence contains a sequence identical orcomplementary to 5′-CAGTTG. (See, SEQ ID NOS:27, 33 & 92-101).Additions, deletions and mismatches may be incorporated into a promoterprimer/promoter provider oligomer's target hybridizing sequenceconfigured to hybridize to a region of SEQ ID NO:91. Representativetarget hybridizing sequences for some of the promoter primer/provideroligomers are shown in upper case lettering with an exemplary promotersequence shown in lower case lettering in Table 1 (T7 Providers/Primer).Also shown in Table 1 are representative target hybridizing sequences(See SEQ ID NOS:19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,47, & 49). These target hybridizing sequences are not limited to thetarget hybridizing sequence of a promoter based amplification oligomers,and can be used as primers or other amplification oligomer types.

Blocker oligomers may be used in a single primer transcriptionassociated amplification reaction. Embodiments of 3′ blocked oligomersinclude those of SEQ ID NOS:50-57 where an the 3′ end of the sequence ispreferably blocked to prevent primer-based nucleic acid synthesis fromoccurring. (See Table 1, Blocker)

TABLE 1 16S rRNA Amplification Oligonucleotides SEQ ID Use NO:Sequence (5′→3′) Primer 1 CACCGAAAAACTTTCCCTACTCAAC Primer 2CTACACCGAAAAACTTTCCCTACTCAAC Primer 3 CCTACACCGAAAAACTTTCCCTACTCAACPrimer 4 CACCGAAAAACTTTCCCTACTC Primer 5 CTACACCGAAAAACTTTCCCTACTCPrimer 6 CCTACACCGAAAAACTTTCCCTACTC Primer 7 GTCTCATCCTACACCGAAAAACPrimer 8 CTATATAGTCTCATCCTACACCG Primer 9 GCTGATACTATATAGTCTCATCCTACACCGPrimer 10 CTATATAGTCTCATCCTACACC Primer 11 GCTGATACTATATAGTCTCATCCTACACCPrimer 12 CTATATAGTCTCATCCTACAC Primer 13CCAACTAGCTGATACTATATAGTCTCATCCTACAC Primer 14 GCTGATACTATATAGTCTCATCCPrimer 15 GTGCGCCACTAATCCACTTC Primer 16 CGTGCGCCACTAATCCACTT Primer 17CGTGCGCCACTAATCCACT T7 Provider*/Primer 18aatttaatacgactcactatagggagaCCTACACAAGAGGACAACA GTTG Target Hybridizing19 CCTACACAAGAGGACAACAGTTG Seq. T7 Provider/Primer 20aatttaatacgactcactatagggagaCCTACACAAGAGGACAACA GTTGG Target Hybridizing21 CCTACACAAGAGGACAACAGTTGG Seq. T7 Provider/Primer 22aatttaatacgactcactatagggagaCACAAGAGGACAACAGTTG GAAACG Target Hybridizing23 CACAAGAGGACAACAGTTGGAAACG Seq. T7 Provider/Primer 24aatttaatacgactcactatagggagaCACAAGAGGACAACAGTTG GAAACGACTarget Hybridizing 25 CACAAGAGGACAACAGTTGGAAACGAC Seq.T7 Provider/Primer 26 aatttaatacgactcactatagggagaAAGAGGACAACAGTTGGAA ACTarget Hybridizing 27 AAGAGGACAACAGTTGGAAAC Seq. T7 Provider/Primer 28aatttaatacgactcactatagggagaGGACAACAGTTGGAAACGA CTGCTAATACTCTTarget Hybridizing 29 GGACAACAGTTGGAAACGACTGCTAATACTCT Seq.T7 Provider/Primer 30 aatttaatacgactcactatagggagaCAACAGTTGGAAACGACTGCTAATACTCT Target Hybridizing 31 CAACAGTTGGAAACGACTGCTAATACTCT Seq.T7 Providcr/Primer 32 aatttaatacgactcactatagggagaCAGTTGGAAACGACTGCTAATACTCT Target Hybridizing 33 CAGTTGGAAACGACTGCTAATACTCT Seq.T7 Provider/Primer 34 aatttaatacgactcactatagggagaGGCGTGCCTAATACATGCAAGTCG Target Hybridizing 35 GGCGTGCCTAATACATGCAAGTCG Seq.T7 Provider/Primer 36 aatttaatacgactcactatagggagaCGTGCCTAATACATGCAAG TCGTarget Hybridizing 37 CGTGCCTAATACATGCAAGTCG Seq. T7 Provider/Primer 38aatttaatacgactcactatagggagaGCCTAATACATGCAAGTCG AAC Target Hybridizing 39GCCTAATACATGCAAGTCGAAC Seq. T7 Provider/Primer 40aatttaatacgactcactatagggagaCCTAATACATGCAAGTCGA ACG Target Hybridizing 41CCTAATACATGCAAGTCGAACG Seq. T7 Provider/Primer 42aatttaatacgactcactatagggagaGCTAGAAGTGGATTAGTGG CGCAC Target Hybridizing43 GCTAGAAGTGGATTAGTGGCGCAC Seq. T7 Provider/Primer 44aatttaatacgactcactatagggagaGGATTAGTGGCGCACGGGT GAG Target Hybridizing 45GGATTAGTGGCGCACGGGTGAG Seq. T7 Provider/Primer 46aatttaatacgactcactatagggagaGGGTGAGTAAGGTATAGTT AATCTGCTarget Hybridizing 47 GGGTGAGTAAGGTATAGTTAATCTGC Seq. T7 Provider/Primer48 aatttaatacgactcactatagggagaGGTGAGTAAGGTATAGTTA ATCTGCCTarget Hybridizing 49 GGTGAGTAAGGTATAGTTAATCTGCC Seq. Blocker 50CAACTGTTGTCCTCTTGTG Blocker 51 CTAGCAAGCTAGAAGCTTC Blocker 52CTAATCCACTTCTAGCAAGC Blocker 53 TCACCCGTGCGCCACTAATC Blocker 54TTAGGCACGCCGCCAG Blocker 55 ATTAGGCACGCCGCCAG Blocker 56GTAGGGCAGATTAACTATAC Blocker 57 CTTGTGTAGGGCAGATTAAC

Some exemplary detection probes useful with the current inventioninclude linear and hairpin oligonucleotides that are from about 10 toabout 70 nucleotides in length and comprise a target hybridizingsequence that is configured to hybridize to either the “+” or the “−”strand of a region of SEQ ID NO:91, or RNA equivalent thereof, whereinthe region of SEQ ID NO:91 is from about nucleotide 14 to aboutnucleotide 226; is from about nucleotide 108 to about nucleotide 226; isfrom about nucleotide 134 to about nucleotide 181; is from aboutnucleotide 159 to about nucleotide 181; is from about nucleotide 159 toabout nucleotide 181 and the probe contains a sequence identical orcomplementary to nucleotides 167 to 181 of SEQ ID NO:91; is from aboutnucleotide 147 to about nucleotide 181; is from about nucleotide 163 toabout nucleotide 181; or is from about nucleotide 163 to aboutnucleotide 181 and the probe contains a sequence identical orcomplementary to nucleotides 167 to 181. (See, SEQ ID NOS:102-106)Optionally, when the detection probe oligo is a molecular torch, thedetection oligo also comprises target closing domains or otherhairpin-forming structure. Additions, deletions and mismatches may beincorporated into a detection probe oligo designed to hybridize to aregion of SEQ ID NO:91. Representative detection probe oligo sequencesare shown in Table 2 as SEQ ID NOS:59-70 (Torch).

For the sequences listed in Table 2, the lowercase letters indicate thenucleotides in the sequence that form part of the closing domain, butarc not part of the target binding sequence. Embodiments of the hairpinprobe oligomers were synthesized with a fluorescent label attached atone end of the sequence and a quencher compound attached at the otherend of the sequence. Some embodiments of hairpin oligomers also includea non-nucleotide linker moiety at selected positions within thesequence. Examples of such embodiments include those that include anabasic 9-carbon (“C9”) linker between residues 6 and 7 of SEQ ID NO.:70, between residues 15 and 16 of SEQ ID NO.: 68, between residues 16and 17 of SEQ ID NO.: 61, between residues 17 and 18 of SEQ ID NO.: 67,between residues 18 and 19 of SEQ ID NO.: 69, between residues 20 and 21of SEQ ID NO.: 66, between residues 21 and 22 of SEQ ID NO.: 63, betweenresidues 23 and 25 of SEQ ID NO.: 62, between residues 24 and 25 of SEQID NO.: 65, between residues 25 and 26 of SEQ ID NOS.: 59 and 64, andbetween residues 26 and 27 of SEQ ID NO.:60. Detection probes may beused with helper probes that are unlabeled and facilitate binding of thelabeled probe to its target as previously described (U.S. Pat. No.5,030,557).

TABLE 2 16S rRNA Probes Use SEQ ID NO: Sequence (5′→3′) Torch 59cGGAGTATAGAGTATTAGCAGTCGTcTCCg Torch 60 GCAGGAGTATAGAGTATTAGCAGTCGcctgcTorch 61 GCAGGAGTATAGAGTAcctGC Torch 62 ccGTGTTAAGCAGGAGTATAGAGcacggTorch 63 ccGTGTTAAGCAGGAGTATAGcacgg Torch 64 cTCCCTACTCAACTTGTGTTAAGCAGGgag Torch 65 cTCCCTACTCAACTTGTGTTAAGCgGGAG Torch 66cTCCCTACTCAACTTGTGTTggGag Torch 67 cTCCCTACTCAACTTGTGggag Torch 68cTCCCTACTCAACTTGggag Torch 69 ccGCTAGAAGCTTCATCGagcgg Torch 70cggaaGCAAGCTAGAAGCTTCcg

Capture probe oligomers may be used in sample preparation to separate C.jejuni, C. lari, and/or C. coli target nucleic acids from other samplecomponents. Exemplary capture probe oligomers include those comprising atarget-specific sequence of SEQ ID NOS:71-74. SEQ ID NOS:71 and 73 areembodiments of capture probes that include the target-specific sequencesof SEQ ID NOS:72 and 74 and a binding partner that can be a nucleic acidor non-nucleic acid binding partner. SEQ ID NOS:72 and 74 each include abinding partner that is a dT.sub.3A.sub.30 polymer (underlined). Table3.

TABLE 3 Capture Oligos. SEQ ID Use NO: Sequence (5′→3′) Target  71GCGTCAGGGTTTCCCCCATTGCGTTTAAAAAAAA Capture AAAAAAAAAAAAAAAAAAAAAATarget  72 GCGTCAGGGTTTCCCCCATTGCG [binding  Capture partner] Target  73GCTTATTCCTTAGGTACCGTCAGTTTAAAAAAAA Capture AAAAAAAAAAAAAAAAAAAAAATarget  74 GCTTATTCCTTAGGTACCGTCAG [binding  Capture partner]

Example 1. Assay Reagents, Equipment and Protocols

The following example describes typical assay reagents, equipment,protocols, conditions and the like used in the real-time TMA experimentsdescribed herein. Unless specified to the contrary, reagent preparation,equipment preparation and assay protocols were performed essentially asset forth below. An ordinarily skilled artisan in possession of thisdisclosure can vary the reagents, conditions, instruments and assaymethods, and such variations are within the scope of this disclosure.

Reagents used in target capture and amplification steps in the examplesdescribed herein generally included one or more of the following. ProbeMatrix Lysis Reagent contained 0.1% (w/v) Lithium Lauryl Sulfate (LLS),20 mM Lithium Succinate, and 1 mM EDTA. Lysis Reagent contained 1% (w/v)LLS, 100 mM Tris, 2.5 mM succinic acid, 10 mM EDTA, and 500 mM lithiumchloride (LiCl) at pH 6.5. Target Capture Reagent contained 300 mMHEPES, 1.88 M lithium chloride, 100 mM EDTA, at pH 6.4, and 250.micro.g/ml of paramagnetic particles (0.7-1.05 micron particles,SERA-MAG.sup.™ MG-CM, Seradyn, Inc., Indianapolis, Ind.) with(dT).sub.14 oligomers covalently bound thereto. Wash Solution used intarget capture contained 10 mM HEPES, 150 mM NaCl, 1 mM EDTA, and 0.1%(w/v) sodium dodecyl sulfate, at pH 7.5. Amplification Reagent was mixedwith other reaction components produce a solution containing 50 mMHEPES, 33 mM KCl, 30 mM MgCl.sub.2, 0.5 mM of each dNTP (dATP, dCTP,dGTP, dTTP), 10 mM ATP, 2 mM CTP, 12.7 mM UTP, 2 mM GTP, at pH 7.7.Amplification oligonucleotides (primers 0.125 pmol/.micro.L, promoterprimers 0.125 pmol/.micro.L, blocker oligonucleotides 0.0125pmol/.micro.L, promoter provider oligonucleotides) 0.125 pmol/.micro.L,and optionally probes 0.3 pmol/.micro.L, may be added to the reactionmixture in the amplification reagent or separate from the amplificationreagent. Enzyme Reagent contained 360 RTU/.micro.l of Moloney murineleukemia virus (MMLV) reverse transcriptase (RT) and about 80PU/.micro.l of T7 RNA polymerase, 75 mM HEPES, 120 mM KCl, 10% TRITON®X-100, 160 mM N-acetyl-L-cysteine, and 1 mM EDTA at pH 7.0, where 1 RTUof RT incorporates 1 nmol of dTTP in 20 min at 37.deg.C., and 1 PU of T7RNA polymerase produces 5 nmol of RNA transcript in 20 min at 37.deg.C.

Equipment and Material generally included: KingFisher® 96 (ThermoElectron, Waltham, Mass.); KingFisher® mL (Thermo Electron, Waltham,Mass.); PTI-FP-2® FluoDia Plate Reader (Photon Technology International,Birmingham, N.J.); Eppendorf® Thermomixer R 022670565 (EppendorfCorporation, Westbury, N.Y.); Hard-Shell Thin-Wall 96-Well Skirted PCRPlates, colored shell/white well, Catalog numbers: HSP-9615, HSP-9625,HSP-9635) (BioRad Hercules, Calif.); KingFisher® 96 tip comb for DWmagnets (Catalog number: 97002534) Thermo Electron, Waltham, Mass.); DW96 plate, V bottom, Polypropylene, sterile 25 pcs/case (Axygen Catalognumber: P-2ML-SQ-C-S; VWR catalog number 47749-874); KingFisher® 96 KFplate (200 microliters) (Catalog number: 97002540); KingFisher® mL tipcomb (Catalog number 97002111); and KingFisher® mL tubes (Catalog number97002121).

The transcription mediated amplification (TMA) reactions usesubstantially the procedures as disclosed in U.S. Pat. Nos. 5,399,491and 5,554,516. Single primer transcription associated amplificationsubstantially use the procedures disclosed in detail in U.S. Pat. No.7,374,885. The TAG amplification method has been disclosed in US Pub.No. 20070281317 A1. The use and detection of signal from AE-labeledprobes to detect hybridization complexes with target sequences use theprocedures already disclosed in detail in U.S. Pat. Nos. 5,283,174;5,656,744; and 5,658,737. The methods for using hairpin probes are wellknown, and include those already disclosed in detail in U.S. Pat. Nos.6,849,412; 6,835,542; 6,534,274; and 6,361,945.

By using various combinations of the herein described oligomers,including amplification oligomers and labeled detection probe oligomers,C. jejuni, C. lari, and/or C. coli target nucleic acids werespecifically detected when the sample contained about 1E2 copies of thetarget nucleic acid. The following examples illustrate some of theembodiments of the disclosure for detection of C. jejuni, C. lari,and/or C. coli target nucleic acid.

Example 2. Design and Initial Testing of C. jejuni, C. Lari, and C. coli16S Oligomer Combination Sets

Amplification and detection oligonucleotides were configured todiscriminately amplify and detect C. jejuni, C. lari, and C. coli targetnucleic acid. Using two overlapping regions of Campylobacter jejuni 16SrRNA corresponding to nucleotides 9-82 and nucleotides 44-226 ofAF393202.1, gi:20378208, (SEQ ID NO:91), several T7 Providers, Blockers,Primers and Torches were configured to hybridize to smaller sequenceswithin these regions of said reference sequence, as is described herein.The target hybridizing sequences of these oligomers also hybridize to aC. jejuni target nucleic acid and a C. coli, a C. lari or a C. coli anda C. lari target nucleic acid. In addition, using a region correspondingto base pairs 341-426 of this same C. jejuni 16S rRNA, several TargetCapture Oligos were designed. (See Tables 1-3). These oligos weredesigned to maximize selective detection of the different strains of C.jejuni, C. lari, and C. coli with decreased potential for crossreactivity with other organisms.

A total of 942 different combinations of T7 Provider, Blocker, Primerand Torch oligos from Tables 1-2 were screened using a master-platescreening assay. Known amounts of C. jejuni target nucleic acids wereamplified using real time single primer transcription associatedamplification. Selecting one oligomer from each of the following groupsproduced the amplification oligomer combinations used in this initialscreen: group 1 (SEQ ID NOS:1-17); group 2 (SEQ ID NOS:18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 46 or 48); group 3 (SEQ IDNOS:50-58); and group 4 (SEQ ID NOS:59-70). A master-plate was set up tocontain 0.5 pM/.micro.L T7 Provider, 0.5 pM/.micro.L Primer, and 0.5pM/.micro.L Blocker in amplification reagent at a final volume of 200.micro.L per reaction well. Each reaction well contained one of theoligo combinations. The master-plate was kept at 20.deg.C.

Primary oligo screening included transferring 10 .micro.L of each oligocombination into a 96-well plate. C. jejuni target nucleic acid(American Type Cell Culture, Manassas, Va., ATCC 33560) was diluted to1E5 copies per/20 .micro.l in amplification reagent and the dilutedtarget was then added to each well of the 96-well plate so that the C.jejuni target nucleic acid was tested at 1E5 copies/reaction. A negativecontrol containing oligos and Amplification mixture without target wasincluded on the plates. The reaction mixtures containing theoligonucleotides, target and amplification reagents were covered toprevent evaporation, incubated 10 min at 60.deg.C. and then cooled to42.deg.C. The enzyme reagent was added next at 10 .micro.L per well andthe reactions were mixed and incubated at 42.deg.C.

Fluorescence was measured at regular time intervals (e.g., every 72 sec)during the amplification reaction for a total of 63 reads. Two colorswere read at each interval. Monitoring was done using a real timefluorescent detection system (e.g., Bio-Rad Laboratory's Opticon.sup.™or Chromo4.sup.™, or a FluoDia® T70 instrument). Real time algorithmsare well known in the art, examples are described in Biochem Biophys ResCommun. 2002 Jun. 7; 294(2):347-53 and Nucleic Acids Research 200432(22):e178. The negative controls were tested to determine backgroundfluorescent signal levels. Raw data were gathered and analyzed tocalculate signal emergence time (TTime) and range relative fluorescentunits (RFUs). Oligo combinations were then grouped based upon TTime withTTime under 23 minutes being considered as the best combinations, TTimesbetween 23 minutes and 29 minutes being considered as adequate oligocombinations and TTimes of 30 or more minutes being unacceptable.Positive criterion depended on the instrument used. For instance,positive criterion was set at 0.05 RFU and was assessed over a 67-minutetime course for the MJ Chromo4, whereas positive criterion was set at1000 RFU and was assessed over a 75-minute time course using a differentplate reader instrument. The oligomer combinations listed in Table 4produced a fluorescent curve with a TTime less than or equal to 23minutes. The oligomer combinations listed in Table 5 produced afluorescent curve with a TTime between 23 and 30 minutes. From Tables 4and 5, a preferred set of oligonucleotide combinations was selected forsecondary screening. In addition to TTime, oligo combinations were alsolooked at to detect design flaws such as the number of mismatchednucleobases compared to Campylobacter species and the degree of burdenfor specificity placed upon each oligonucleotide in a combination. Otherfactors impacting the selection of oligo combinations for secondaryscreening included poor oligo compatibility and poor performance inamplifying and/or detecting target.

TABLE 4 Campylobacter Oligomer Combinations Less than 23 minutes ComboNo. SEQ ID NOS. 1 1, 48, 53, 59 2 1, 18, 56, 59 3 1, 18, 57, 59 4 1, 20,56, 59 5 1, 20, 57, 59 6 1, 46, 53, 60 7 1, 48, 53, 60 8 1, 18, 56, 60 91, 18, 57, 60 10 1, 20, 56, 60 11 1, 20, 57, 60 12 1, 46, 53, 61 13 1,48, 53, 61 14 1, 18, 56, 61 15 1, 18, 57, 61 16 1, 20, 56, 61 17 1, 20,57, 61 18 1, 48, 53, 62 19 1, 18, 56, 62 20 1, 18, 57, 62 21 1, 20, 56,62 22 1, 20, 57, 62 23 1, 18, 56, 63 24 1, 18, 57, 63 25 1, 20, 56, 6326 1, 20, 57, 63 27 2, 46, 53, 60 28 2, 18, 56, 60 29 2, 20, 56, 60 302, 20, 57, 60 31 2, 20, 56, 61 32 2, 20, 57, 61 33 4, 48, 53, 59 34 4,18, 56, 59 35 4, 18, 57, 59 36 4, 20, 56, 59 37 4, 20, 57, 59 38 4, 46,53, 60 39 4, 48, 53, 60 40 4, 18, 56, 60 41 4, 18, 57, 60 42 4, 20, 56,60 43 4, 20, 57, 60 44 4, 46, 53, 61 45 4, 48, 53, 61 46 4, 18, 56, 6147 4, 18, 57, 61 48 4, 20, 56, 61 49 4, 20, 57, 61 50 4, 48, 53, 62 514, 18, 56, 62 52 4, 18, 57, 62 53 4, 20, 56, 62 54 4, 20, 57, 62 55 4,18, 56, 63 56 4, 18, 57, 63 57 4, 20, 56, 63 58 4, 20, 57, 63 59 5, 46,53, 59 60 5, 18, 56, 59 61 5, 18, 57, 59 62 5, 20, 56, 59 63 5, 20, 57,59 64 5, 46, 53, 60 65 5, 48, 53, 60 66 5, 18, 56, 60 67 5, 18, 57, 6068 5, 20, 56, 60 69 5, 20, 57, 60 70 5, 46, 53, 61 71 5, 18, 56, 61 725, 18, 57, 61 73 5, 20, 56, 61 74 5, 20, 57, 61 75 5, 18, 56, 62 76 5,20, 56, 62 77 5, 20, 57, 62 78 5, 20, 56, 63 79 5, 20, 57, 63 80 6, 46,53, 60 81 6, 48, 53, 60 82 7, 20, 57, 59 83 7, 42, 51, 60 84 7, 44, 52,60 85 7, 46, 53, 60 86 7, 48, 53, 60 87 7, 18, 56, 60 88 7, 18, 57, 6089 7, 20, 56, 60 90 7, 20, 57, 60 91 7, 46, 53, 61 92 7, 48, 53, 61 937, 18, 56, 61 94 7, 18, 57, 61 95 7, 20, 56, 61 96 7, 20, 57, 61 97 7,28, 50, 61 98 7, 30, 50, 61 99 7, 32, 50, 61 100 7, 20, 56, 62 101 7,20, 57, 62 102 7, 24, 57, 62 103 7, 28, 50, 62 104 7, 30, 50, 62 105 7,32, 50, 62 106 7, 46, 53, 63 107 7, 48, 53, 63 108 7, 18, 56, 63 109 7,18, 57, 63 110 7, 20, 56, 63 111 7, 20, 57, 63 112 7, 28, 50, 63 113 7,30, 50, 63 114 7, 32, 50, 63 115 7, 20, 57, 65 116 7, 28, 50, 65 117 7,30, 50, 65 118 7, 32, 50, 65 119 7, 46, 53, 66 120 7, 48, 53, 66 121 7,18, 56, 66 122 7, 18, 57, 66 123 7, 20, 56, 66 124 7, 20, 57, 66 125 7,30, 50, 66 126 7, 32, 50, 66 127 7, 20, 57, 67 128 7, 28, 50, 67 129 7,30, 50, 67 130 7, 32, 50, 67 131 7, 18, 57, 68 132 7, 32, 50, 68 133 7,20, 56, 68 134 7, 20, 57, 68 135 7, 28, 50, 68 136 7, 30, 56, 68 137 8,20, 56, 59 138 8, 20, 57, 59 139 8, 46, 53, 60 140 8, 48, 53, 60 141 8,18, 56, 60 142 8, 18, 57, 60 143 8, 20, 56, 60 144 8, 20, 57, 60 145 8,46, 53, 61 146 8, 48, 53, 61 147 8, 18, 56, 61 148 8, 18, 57, 61 149 8,20, 56, 61 150 8, 20, 57, 61 151 8, 28, 50, 61 152 8, 30, 50, 61 153 8,32, 50, 61 154 8, 18, 57, 62 155 8, 20, 56, 62 156 8, 20, 57, 62 157 8,24, 57, 62 158 8, 28, 50, 62 159 8, 30, 50, 62 160 8, 32, 50, 62 161 8,18, 56, 63 162 8, 18, 57, 63 163 8, 20, 56, 63 164 8, 20, 57, 63 165 8,24, 57, 63 166 8, 28, 50, 63 167 8, 30, 50, 63 168 8, 32, 50, 63 169 8,20, 56, 65 170 8, 20, 57, 65 171 8, 28, 50, 65 172 8, 30, 50, 65 173 8,32, 50, 65 174 8, 18, 56, 66 175 8, 18, 57, 66 176 8, 20, 56, 66 177 8,20, 57, 66 178 8, 28, 50, 66 179 8, 30, 50, 66 180 8, 32, 50, 66 181 8,20, 56, 67 182 8, 20, 57, 67 183 8, 28, 50, 67 184 8, 30, 50, 67 185 8,32, 50, 67 186 8, 18, 57, 68 187 8, 20, 56, 68 188 8, 20, 57, 68 189 8,28, 50, 68 190 8, 30, 50, 68 191 8, 32, 50, 68 192 9, 48, 53, 59 193 9,18, 56, 59 194 9, 18, 57, 59 195 9, 20, 56, 59 196 9, 20, 57, 59 197 9,42, 51, 60 198 9, 46, 53, 60 199 9, 48, 53, 60 200 9, 18, 56, 60 201 9,18, 57, 60 202 9, 20, 56, 60 203 9, 20, 57, 60 204 9, 42, 51, 61 205 9,46, 53, 61 206 9, 48, 53, 61 207 9, 18, 56, 61 208 9, 18, 57, 61 209 9,20, 56, 61 210 9, 20, 57, 61 211 9, 28, 50, 61 212 9, 30, 50, 61 213 9,32, 50, 61 214 9, 48, 53, 62 215 9, 18, 56, 62 216 9, 18, 57, 62 217 9,20, 56, 62 218 9, 20, 57, 62 219 9, 32, 50, 62 220 9, 46, 53, 63 221 9,48, 53, 63 222 9, 18, 56, 63 223 9, 18, 57, 63 224 9, 20, 56, 63 225 9,20, 57, 63 226 9, 24, 57, 63 227 9, 30, 50, 63 228 9, 32, 50, 63 229 9,48, 53, 65 230 9, 20, 56, 65 231 9, 20, 57, 65 232 9, 28, 50, 65 233 9,30, 50, 65 234 9, 32, 50, 65 235 9, 46, 53, 66 236 9, 48, 53, 66 237 9,18, 56, 66 238 9, 18, 57, 66 239 9, 20, 56, 66 240 9, 20, 57, 66 241 9,28, 50, 66 242 9, 30, 50, 66 243 9, 32, 50, 66 244 9, 20, 56, 67 245 9,20, 57, 67 246 9, 28, 50, 67 247 9, 30, 50, 67 248 9, 32, 50, 67 249 9,46, 53, 68 250 9, 48, 53, 68 251 9, 18, 56, 68 252 9, 18, 57, 68 253 9,20, 56, 68 254 9, 20, 57, 68 255 9, 28, 50, 68 256 9, 30, 50, 68 257 9,32, 50, 68 258 10, 20, 56, 59 259 10, 20, 57, 59 260 10, 46, 53, 60 26110, 48, 53, 60 262 10, 18, 56, 60 263 10, 18, 57, 60 264 10, 20, 56, 60265 10, 20, 57, 60 266 10, 46, 53, 61 267 10, 48, 53, 61 268 10, 18, 56,61 269 10, 18, 57, 61 270 10, 20, 56, 61 271 10, 20, 57, 61 272 10, 28,50, 61 273 10, 30, 50, 61 274 10, 32, 50, 61 275 10, 18, 57, 62 276 10,20, 56, 62 277 10, 20, 57, 62 278 10, 24, 57, 62 279 10, 28, 50, 62 28010, 30, 50, 62 281 10, 32, 50, 62 282 10, 18, 56, 63 283 10, 18, 57, 63284 10, 20, 56, 63 285 10, 20, 57, 63 286 10, 30, 50, 63 287 10, 32, 50,63 288 10, 20, 56, 65 289 10, 20, 57, 65 290 10, 28, 50, 65 291 10, 30,50, 65 292 10, 32, 50, 65 293 10, 18, 56, 66 294 10, 18, 57, 66 295 10,20, 56, 66 296 10, 20, 57, 66 297 10, 28, 50, 66 298 10, 30, 50, 66 29910, 32, 50, 66 300 10, 18, 57, 67 301 10, 20, 56, 67 302 10, 20, 57, 67303 10, 28, 50, 67 304 10, 30, 50, 67 305 10, 32, 50, 67 306 10, 18, 57,68 307 10, 20, 56, 68 308 10, 20, 57, 68 309 10, 28, 50, 68 310 10, 30,50, 68 311 10, 32, 50, 68 312 11, 48, 53, 59 313 11, 18, 57, 59 314 11,20, 56, 59 315 11, 20, 57, 59 316 11, 42, 51, 60 317 11, 44, 52, 60 31811, 46, 53, 60 319 11, 48, 53, 60 320 11, 18, 56, 60 321 11, 18, 57, 60322 11, 20, 56, 60 323 11, 20, 57, 60 324 11, 46, 53, 61 325 11, 48, 53,61 326 11, 18, 56, 61 327 11, 18, 57, 61 328 11, 20, 56, 61 329 11, 20,57, 61 330 11, 24, 57, 61 331 11, 28, 50, 61 332 11, 30, 50, 61 333 11,32, 50, 61 334 11, 48, 53, 62 335 11, 18, 57, 62 336 11, 20, 56, 62 33711, 20, 57, 62 338 11, 22, 57, 62 339 11, 32, 50, 62 340 11, 46, 53, 63341 11, 48, 53, 63 342 11, 18, 56, 63 343 11, 18, 57, 63 344 11, 20, 56,63 345 11, 20, 57, 63 346 11, 48, 53, 65 347 11, 18, 56, 65 348 11, 20,56, 65 349 11, 20, 57, 65 350 11, 28, 50, 65 351 11, 30, 50, 65 352 11,32, 50, 65 353 11, 46, 53, 66 354 11, 48, 53, 66 355 11, 18, 56, 66 35611, 18, 57, 66 357 11, 20, 56, 66 358 11, 20, 57, 66 359 11, 28, 50, 66360 11, 30, 50, 66 361 11, 32, 50, 66 362 11, 48, 53, 67 363 11, 18, 57,67 364 11, 20, 56, 67 365 11, 20, 57, 67 366 11, 28, 50, 67 367 11, 30,50, 67 368 11, 32, 50, 67 369 11, 48, 53, 68 370 11, 18, 57, 68 371 11,20, 56, 68 372 11, 20, 57, 68 373 11, 28, 50, 68 374 11, 30, 50, 68 37511, 32, 50, 68 376 12, 48, 53, 59 377 12, 18, 57, 59 378 12, 20, 56, 59379 12, 20, 57, 59 380 12, 42, 51, 60 381 12, 44, 52, 60 382 12, 46, 53,60 383 12, 48, 53, 60 384 12, 18, 56, 60 385 12, 18, 57, 60 386 12, 20,56, 60 387 12, 20, 57, 60 388 12, 46, 53, 61 389 12, 48, 53, 61 390 12,18, 56, 61 391 12, 18, 57, 61 392 12, 20, 56, 61 393 12, 20, 57, 61 39412, 28, 50, 61 395 12, 30, 50, 61 396 12, 32, 50, 61 397 12, 48, 53, 62398 12, 18, 57, 62 399 12, 20, 56, 62 400 12, 20, 57, 62 401 12, 46, 53,63 402 12, 48, 53, 63 403 12, 18, 56, 63 404 12, 18, 57, 63 405 12, 20,56, 63 406 12, 20, 57, 63 407 12, 20, 56, 65 408 12, 20, 57, 65 409 12,28, 50, 65 410 12, 30, 50, 65 411 12, 32, 50, 65 412 12, 18, 56, 66 41312, 18, 57, 66 414 12, 20, 56, 66 415 12, 20, 57, 66 416 12, 28, 50, 66417 12, 30, 50, 66 418 12, 32, 50, 66 419 12, 20, 56, 67 420 12, 20, 57,67 421 12, 28, 50, 67 422 12, 30, 50, 67 423 12, 32, 50, 67 424 12, 18,57, 68 425 12, 20, 56, 68 426 12, 20, 57, 68 427 12, 28, 50, 68 428 12,30, 50, 68 429 12, 32, 50, 68 430 13, 18, 56, 59 431 13, 18, 57, 59 43213, 20, 56, 59 433 13, 20, 57, 59 434 13, 18, 56, 60 435 13, 18, 57, 60436 13, 20, 56, 60 437 13, 18, 57, 61 438 13, 20, 56, 61 439 13, 28, 50,61 440 13, 30, 50, 61 441 13, 32, 50, 61 442 13, 46, 53, 62 443 13, 48,53, 62 444 13, 18, 56, 62 445 13, 18, 57, 62 446 13, 20, 56, 62 447 13,32, 50, 62 448 13, 46, 53, 63 449 13, 48, 53, 63 450 13, 18, 56, 63 45113, 18, 57, 63 452 13, 20, 56, 63 453 13, 20, 57, 63 454 13, 20, 56, 65455 13, 20, 57, 65 456 13, 28, 50, 65 457 13, 30, 50, 65 458 13, 32, 50,65 459 13, 20, 56, 66 460 13, 20, 57, 66 461 13, 28, 50, 66 462 13, 30,50, 66 463 13, 32, 50, 66 464 13, 20, 56, 67 465 13, 20, 57, 67 466 13,28, 50, 67 467 13, 30, 50, 67 468 13, 32, 50, 67 469 13, 20, 57, 68 47013, 28, 50, 68 471 13, 30, 50, 68 472 13, 32, 50, 68 473 14, 48, 53, 59474 14, 18, 56, 59 475 14, 18, 57, 59 476 14, 20, 56, 59 477 14, 20, 57,59 478 14, 46, 53, 60 479 14, 48, 53, 60 480 14, 18, 56, 60 481 14, 18,57, 60 482 14, 20, 56, 60 483 14, 20, 57, 60 484 14, 48, 53, 61 485 14,18, 57, 61 486 14, 20, 57, 61 487 14, 28, 50, 61 488 14, 30, 50, 61 48914, 32, 50, 61 490 14, 46, 53, 62 491 14, 48, 53, 62 492 14, 18, 56, 62493 14, 18, 57, 62 494 14, 20, 56, 62 495 14, 46, 53, 63 496 14, 48, 53,63 497 14, 18, 56, 63 498 14, 18, 57, 63 499 14, 20, 56, 63 500 14, 20,57, 63 501 14, 28, 50, 65 502 14, 30, 50, 65 503 14, 32, 50, 65 504 14,48, 53, 66 505 14, 18, 57, 66 506 14, 28, 50, 66 507 14, 30, 50, 66 50814, 32, 50, 66 509 14, 48, 53, 67 510 14, 18, 57, 67 511 14, 28, 50, 67512 14, 30, 50, 67 513 14, 32, 50, 67 514 14, 20, 56, 68 515 14, 20, 57,68 516 14, 30, 50, 68 517 14, 32, 50, 68 518 15, 34, 54, 69 519 15, 34,55, 69 520 15, 36, 54, 69 521 15, 38, 54, 69 522 15, 40, 54, 69 523 15,34, 54, 70 524 15, 34, 55, 70 525 15, 36, 54, 70 526 15, 36, 55, 70 52715, 38, 54, 70 528 15, 38, 55, 70 529 15, 40, 54, 70 530 15, 40, 55, 70531 16, 34, 54, 69 532 16, 36, 54, 69 533 16, 38, 54, 69 534 16, 40, 54,69 535 16, 40, 55, 69 536 16, 34, 54, 70 537 16, 34, 55, 70 538 16, 36,54, 70 539 16, 36, 55, 70 540 16, 38, 54, 70 541 16, 38, 55, 70 542 16,40, 54, 70 543 16, 40, 55, 70 544 17, 34, 54, 69 545 17, 36, 54, 69 54617, 38, 54, 69 547 17, 40, 54, 69 548 17, 40, 55, 69 549 17, 34, 54, 70550 17, 34, 55, 70 551 17, 36, 54, 70 552 17, 36, 55, 70 553 17, 38, 54,70 554 17, 38, 55, 70 555 17, 40, 54, 70 556 17, 40, 55, 70

TABLE 5 Campylobacter Oligomer Combinations 23-29 minutes Combo No. SEQID NOS. 557 1, 46, 53, 59 558 1, 42, 51, 60 559 1, 44, 52, 60 560 1, 22,57, 61 561 1, 24, 57, 61 562 1, 46, 53, 62 563 1, 22, 57, 62 564 1, 24,57, 62 565 1, 46, 53, 63 566 1, 48, 53, 63 567 1, 22, 57, 63 568 1, 24,57, 63 569 2, 42, 51, 59 570 2, 46, 53, 59 571 2, 48, 53, 59 572 2, 18,56, 59 573 2, 18, 57, 59 574 2, 20, 56, 59 575 2, 20, 57, 59 576 2, 48,53, 60 577 2, 18, 57, 60 578 2, 46, 53, 61 579 2, 18, 56, 61 580 2, 18,57, 61 581 2, 22, 57, 61 582 2, 24, 57, 61 583 2, 46, 53, 62 584 2, 18,56, 62 585 2, 18, 57, 62 586 2, 20, 56, 62 587 2, 20, 57, 62 588 2, 22,57, 62 589 2, 24, 57, 62 590 2, 46, 53, 63 591 2, 18, 56, 63 592 2, 18,57, 63 593 2, 20, 56, 63 594 2, 20, 57, 63 595 2, 22, 57, 63 596 2, 24,57, 63 597 3, 46, 53, 59 598 3, 18, 56, 59 599 3, 18, 57, 59 600 3, 20,56, 59 601 3, 20, 57, 59 602 3, 44, 52, 60 603 3, 46, 53, 60 604 3, 48,53, 60 605 3, 18, 56, 60 606 3, 18, 57, 60 607 3, 20, 56, 60 608 3, 20,57, 60 609 3, 18, 56, 61 610 3, 18, 57, 61 611 3, 20, 56, 61 612 3, 20,57, 61 613 3, 22, 57, 61 614 3, 24, 57, 61 615 3, 18, 56, 62 616 3, 18,57, 62 617 3, 20, 57, 62 618 3, 22, 57, 62 619 3, 24, 57, 62 620 3, 22,57, 63 621 3, 24, 57, 63 622 4, 46, 53, 59 623 4, 22, 57, 61 624 4, 24,57, 61 625 4, 46, 53, 62 626 4, 22, 57, 62 627 4, 24, 57, 62 628 4, 46,53, 63 629 4, 48, 53, 63 630 4, 22, 57, 63 631 4, 24, 57, 63 632 5, 48,53, 59 633 5, 22, 57, 61 634 5, 24, 57, 61 635 5, 46, 53, 62 636 5, 18,57, 62 637 5, 22, 57, 62 638 5, 24, 57, 62 639 5, 46, 53, 63 640 5, 48,53, 63 641 5, 18, 56, 63 642 5, 18, 57, 63 643 5, 22, 57, 63 644 5, 24,57, 63 645 6, 46, 53, 59 646 6, 48, 53, 59 647 6, 18, 56, 59 648 6, 18,57, 59 649 6, 20, 56, 59 650 6, 20, 57, 59 651 6, 18, 56, 60 652 6, 18,57, 60 653 6, 20, 56, 60 654 6, 20, 57, 60 655 6, 46, 53, 61 656 6, 18,56, 61 657 6, 18, 57, 61 658 6, 20, 56, 61 659 6, 20, 57, 61 660 6, 22,57, 61 661 6, 24, 57, 61 662 6, 46, 53, 62 663 6, 48, 53, 62 664 6, 18,56, 62 665 6, 18, 57, 62 666 6, 20, 56, 62 667 6, 20, 57, 62 668 6, 22,57, 62 669 6, 24, 57, 62 670 6, 46, 53, 63 671 6, 18, 56, 63 672 6, 18,57, 63 673 6, 20, 56, 63 674 6, 20, 57, 63 675 6, 22, 57, 63 676 6, 24,57, 63 677 7, 46, 53, 59 678 7, 48, 53, 59 679 7, 18, 56, 59 680 7, 18,57, 59 681 7, 20, 56, 59 682 7, 42, 51, 61 683 7, 44, 52, 61 684 7, 22,57, 61 685 7, 24, 57, 61 686 7, 46, 53, 62 687 7, 48, 53, 62 688 7, 18,56, 62 689 7, 18, 57, 62 690 7, 22, 57, 62 691 7, 22, 57, 63 692 7, 24,57, 63 693 7, 22, 57, 64 694 7, 24, 57, 64 695 7, 46, 53, 65 696 7, 48,53, 65 697 7, 18, 56, 65 698 7, 18, 57, 65 699 7, 20, 56, 65 700 7, 22,57, 65 701 7, 24, 57, 65 702 7, 22, 57, 66 703 7, 24, 57, 66 704 7, 28,50, 66 705 7, 46, 53, 67 706 7, 48, 53, 67 707 7, 18, 56, 67 708 7, 18,57, 67 709 7, 20, 56, 67 710 7, 22, 57, 67 711 7, 24, 57, 67 712 7, 46,53, 68 713 7, 48, 53, 68 714 7, 18, 56, 68 715 7, 22, 57, 68 716 7, 24,57, 68 717 8, 46, 53, 59 718 8, 18, 56, 59 719 8, 18, 57, 59 720 8, 22,57, 61 721 8, 24, 57, 61 722 8, 46, 53, 62 723 8, 18, 56, 62 724 8, 22,57, 62 725 8, 46, 53, 63 726 8, 48, 53, 63 727 8, 22, 57, 63 728 8, 22,57, 64 729 8, 24, 57, 64 730 8, 18, 56, 65 731 8, 18, 57, 65 732 8, 22,57, 65 733 8, 24, 57, 65 734 8, 46, 53, 66 735 8, 48, 53, 66 736 8, 22,57, 66 737 8, 24, 57, 66 738 8, 18, 56, 67 739 8, 18, 57, 67 740 8, 22,57, 67 741 8, 24, 57, 67 742 8, 46, 53, 68 743 8, 18, 56, 68 744 8, 22,57, 68 745 8, 24, 57, 68 746 9, 46, 53, 59 747 9, 22, 57, 61 748 9, 24,57, 61 749 9, 46, 53, 62 750 9, 22, 57, 62 751 9, 24, 57, 62 752 9, 28,50, 62 753 9, 30, 50, 62 754 9, 22, 57, 63 755 9, 28, 50, 63 756 9, 22,57, 64 757 9, 24, 57, 64 758 9, 46, 53, 65 759 9, 18, 56, 65 760 9, 18,57, 65 761 9, 22, 57, 65 762 9, 24, 57, 65 763 9, 22, 57, 66 764 9, 24,57, 66 765 9, 46, 53, 67 766 9, 48, 53, 67 767 9, 18, 56, 67 768 9, 18,57, 67 769 9, 22, 57, 67 770 9, 24, 57, 67 771 9, 22, 57, 68 772 9, 24,57, 68 773 10, 18, 57, 59 774 10, 22, 57, 61 775 10, 24, 57, 61 776 10,48, 53, 62 777 10, 18, 56, 62 778 10, 22, 57, 62 779 10, 46, 53, 63 78010, 48, 53, 63 781 10, 22, 57, 63 782 10, 24, 57, 63 783 10, 28, 50, 63784 10, 22, 57, 64 785 10, 24, 57, 64 786 10, 18, 56, 65 787 10, 18, 57,65 788 10, 22, 57, 65 789 10, 24, 57, 65 790 10, 46, 53, 66 791 10, 48,53, 66 792 10, 22, 57, 66 793 10, 24, 57, 66 794 10, 48, 53, 67 795 10,18, 56, 67 796 10, 22, 57, 67 797 10, 24, 57, 67 798 10, 48, 53, 68 79910, 18, 56, 68 800 10, 22, 57, 68 801 10, 24, 57, 68 802 11, 46, 53, 59803 11, 18, 56, 59 804 11, 42, 51, 61 805 11, 22, 57, 61 806 11, 46, 53,62 807 11, 18, 56, 62 808 11, 24, 57, 62 809 11, 28, 50, 62 810 11, 30,50, 62 811 11, 42, 51, 63 812 11, 22, 57, 63 813 11, 24, 57, 63 814 11,28, 50, 63 815 11, 30, 50, 63 816 11, 32, 50, 63 817 11, 24, 57, 64 81811, 42, 51, 65 819 11, 46, 53, 65 820 11, 18, 57, 65 821 11, 22, 57, 65822 11, 24, 57, 65 823 11, 22, 57, 66 824 11, 24, 57, 66 825 11, 46, 53,67 826 11, 18, 56, 67 827 11, 22, 57, 67 828 11, 24, 57, 67 829 11, 46,53, 68 830 11, 18, 56, 68 831 11, 22, 57, 68 832 11, 24, 57, 68 833 12,46, 53, 59 834 12, 18, 56, 59 835 12, 42, 51, 61 836 12, 44, 52, 61 83712, 22, 57, 61 838 12, 24, 57, 61 839 12, 46, 53, 62 840 12, 18, 56, 62841 12, 22, 57, 62 842 12, 24, 57, 62 843 12, 28, 50, 62 844 12, 30, 50,62 845 12, 32, 50, 62 846 12, 42, 51, 63 847 12, 22, 57, 63 848 12, 24,57, 63 849 12, 24, 57, 64 850 12, 48, 53, 65 851 12, 18, 56, 65 852 12,18, 57, 65 853 12, 22, 57, 65 854 12, 24, 57, 65 855 12, 46, 53, 66 85612, 48, 53, 66 857 12, 22, 57, 66 858 12, 24, 57, 66 859 12, 46, 53, 67860 12, 48, 53, 67 861 12, 18, 56, 67 862 12, 18, 57, 67 863 12, 22, 57,67 864 12, 24, 57, 67 865 12, 48, 53, 68 866 12, 18, 56, 68 867 12, 22,57, 68 868 12, 24, 57, 68 869 13, 20, 57, 60 870 13, 18, 56, 61 871 13,20, 57, 61 872 13, 22, 57, 61 873 13, 24, 57, 61 874 13, 22, 57, 62 87513, 24, 57, 62 876 13, 28, 50, 62 877 13, 30, 50, 62 878 13, 22, 57, 63879 13, 24, 57, 63 880 13, 30, 50, 63 881 13, 32, 50, 63 882 13, 22, 57,64 883 13, 24, 57, 64 884 13, 18, 56, 65 885 13, 18, 57, 65 886 13, 22,57, 65 887 13, 24, 57, 65 888 13, 18, 56, 66 889 13, 18, 57, 66 890 13,22, 57, 66 891 13, 24, 57, 66 892 13, 18, 56, 67 893 13, 18, 57, 67 89413, 22, 57, 67 895 13, 24, 57, 67 896 13, 18, 56, 68 897 13, 18, 57, 68898 13, 20, 56, 68 899 13, 22, 57, 68 900 13, 24, 57, 68 901 14, 46, 53,59 902 14, 46, 53, 61 903 14, 18, 56, 61 904 14, 20, 56, 61 905 14, 22,57, 61 906 14, 24, 57, 61 907 14, 22, 57, 62 908 14, 24, 57, 62 909 14,28, 50, 62 910 14, 30, 50, 62 911 14, 32, 50, 62 912 14, 22, 57, 63 91314, 24, 57, 63 914 14, 32, 50, 63 915 14, 22, 57, 64 916 14, 24, 57, 64917 14, 46, 53, 65 918 14, 48, 53, 65 919 14, 18, 56, 65 920 14, 18, 57,65 921 14, 20, 56, 65 922 14, 20, 57, 65 923 14, 22, 57, 65 924 14, 24,57, 65 925 14, 46, 53, 66 926 14, 18, 56, 66 927 14, 20, 56, 66 928 14,20, 57, 66 929 14, 22, 57, 66 930 14, 24, 57, 66 931 14, 46, 53, 67 93214, 18, 56, 67 933 14, 20, 56, 67 934 14, 20, 57, 67 935 14, 22, 57, 67936 14, 24, 57, 67 937 14, 46, 53, 68 938 14, 48, 53, 68 939 14, 18, 56,68 940 14, 18, 57, 68 941 14, 22, 57, 68 942 14, 24, 57, 68

A secondary screening was then performed on 22 oligonucleotide setsbased on the initial screening. The oligos selected for secondaryscreening are as follows: T7 Provider SEQ ID NOS: 18, 20, 24, 28, 30, 32and 48; Blocker SEQ ID NOS: 50, 53, 56 and 57; Primer SEQ ID NOS: 4, 7and 12; and Torch SEQ ID NOS: 62, 63, 65, 66, 67 and 68. (See Table 6).These sets of oligos identified for secondary screening were selectedbased upon early emergence times as well as signal and curve shape andother favorable factors.

TABLE 6 Amplification Oligonucleotide Combinations for SecondaryScreening Initial Oligo Screening Combination T7 Provider Blocker PrimerTorch TTime Number (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:)(minutes) 2-1 SEQ ID NO: 18 SEQ ID NO: 56 SEQ ID NO: 4  SEQ ID NO: 6321.9 2-2 SEQ ID NO: 18 SEQ ID NO: 56 SEQ ID NO: 7  SEQ ID NO: 63 19.62-3 SEQ ID NO: 18 SEQ ID NO: 56 SEQ ID NO: 12 SEQ ID NO: 63 19.0 2-4 SEQID NO: 32 SEQ ID NO: 50 SEQ ID NO: 7  SEQ ID NO: 63 19.3 2-5 SEQ ID NO:32 SEQ ID NO: 50 SEQ ID NO: 12 SEQ ID NO: 63 33.0* 2-6 SEQ ID NO: 18 SEQID NO: 56 SEQ ID NO: 7  SEQ ID NO: 68 24.2 2-7 SEQ ID NO: 18 SEQ ID NO:56 SEQ ID NO: 12 SEQ ID NO: 68 24.3 2-8 SEQ ID NO: 32 SEQ ID NO: 50 SEQID NO: 7  SEQ ID NO: 68 18.3 2-9 SEQ ID NO: 32 SEQ ID NO: 50 SEQ ID NO:12 SEQ ID NO: 68 20.2 2-10 SEQ ID NO: 48 SEQ ID NO: 53 SEQ ID NO: 4  SEQID NO: 63 23.7 2-11 SEQ ID NO: 48 SEQ ID NO: 53 SEQ ID NO: 7  SEQ ID NO:63 20.4 2-12 SEQ ID NO: 48 SEQ ID NO: 53 SEQ ID NO: 12 SEQ ID NO: 6317.7 2-13 SEQ ID NO: 48 SEQ ID NO: 53 SEQ ID NO: 12 SEQ ID NO: 68 28.0*2-14 SEQ ID NO: 28 SEQ ID NO: 50 SEQ ID NO: 7  SEQ ID NO: 68 18.8 2-15SEQ ID NO: 30 SEQ ID NO: 50 SEQ ID NO: 7  SEQ ID NO: 68 18.7 2-16 SEQ IDNO: 48 SEQ ID NO: 53 SEQ ID NO: 7  SEQ ID NO: 68 23.4 2-17 SEQ ID NO: 24SEQ ID NO: 57 SEQ ID NO: 7  SEQ ID NO: 68 25.8* 2-18 SEQ ID NO: 24 SEQID NO: 57 SEQ ID NO: 12 SEQ ID NO: 68 26.3* 2-19 SEQ ID NO: 32 SEQ IDNO: 50 SEQ ID NO: 4  SEQ ID NO: 63 assayed in 2^(nd) screen only 2-20SEQ ID NO: 32 SEQ ID NO: 50 SEQ ID NO: 7  SEQ ID NO: 65 18.5 2-21 SEQ IDNO: 32 SEQ ID NO: 50 SEQ ID NO: 7  SEQ ID NO: 66 16.0 2-22 SEQ ID NO: 32SEQ ID NO: 50 SEQ ID NO: 7  SEQ ID NO: 67 17.4 *Combinations chosen forsecondary screening with TTimes higher than 23 minutes. Thesecombinations comprise two or three oligo members that performed well inother combinations.

Example 3. Secondary Screening of Amplification Oligomer Sets:Selectivity for C. jejuni, C. Lari, and C. coli Target Nucleic Acids

The amplification oligomer combinations of Table 6 were further testedfor sensitivity to C. jejuni, C. coli and C. lari target nucleic acid,while discriminating from C. upsaliensis and C. fetus. Each combinationwas tested using 0, 1E2, 1E3 and 1E4 copies of C. jejuni, C. lari, C.coli, C. fetus, or C. upsaliensis 16S rRNA. Amplification oligos werepresent at 5.0 pM T7 Provider, 0.5 pM Blocker, 5.0 pM Primer and 4.0 pMTorch. Positive criterion was 1000 RFU using a PTI plate reader.Amplification oligomer combinations were able to amplify as few as 1E2copies of C. jejuni, C. lari, or C. coli target nucleic acid. TTimeseither emerged at very low levels or very late in the amplificationreaction for C. fetus ssp fetus, C. fetus ssp venerealis and C.upsaliensis. Table 7. Oligo combination numbers (2-8) and (2-22) wereselected for continued screening because in wells comprising theseoligomer combinations in this secondary screening assay neither of theC. fetus species nor the C. upsaliensis species reacted and there wasvery good detection of C. jejuni, C. coli, and C. lari.

TABLE 7 Secondary Screening: Selectivity Screening Results. Oligo C.jejuni; C. fetus, Combination C. coli; C. fetus Number and C. lari sspvenerealis C. upsaliensis Neg (from Table 6) detection detectiondetection Control 2-1 (+) (+) (+) Low Negative 2-2 (+) (+) (+) LowNegative 2-3 (+) (+) Negative Negative 2-4 (+) (+) (+) Low Negative 2-5(+) (+) Negative Negative 2-6 (+) Low Slope Negative Negative Negative2-7 (+) (+) (+) Low (+) Low 2-8 (+) Negative Negative Negative 2-9 (+)(+) (+) Very Low (+) Low 2-10 (+) (+) (+) Very Low Negative 2-11 (+) (+)(+) Very Low Negative 2-12 (+) (+) (+) Very Low Negative 2-13 (+)Negative (+) Very Low Negative 2-14 (+) Negative (+) Low Negative 2-15(+) Negative (+) Low Negative 2-16 (+) Negative (+) Low Negative 2-17(+) Negative (+) Low Negative 2-18 (+) Negative (+) Low Negative 2-19(+) (+) (+) Low Negative 2-20 (+) (+) (+) Low Negative 2-21 (+) (+) LowNegative Negative 2-22 (+) Negative Negative Negative

An additional screen was performed using oligo combination from Table 6to identify torches with sensitivity towards C jejuni. C. lari, and C.coli and discrimination against C. upsaliensis and C. fetus. Positivecriterion was set at 0.05 RFU on a MJ Chrom4 instrument. Theseoligonucleotide sets were tested against 1E5 copies of target organismper reaction. Results of this additional screen indicated that oligocombinations comprising Torch oligonucleotides SEQ ID NOS:63 & 65cross-react with C. fetus, while those comprising Torch SEQ ID NO:66 hadonly a very low signal to C. fetus. Oligo combinations comprising SEQ IDNOS:67-68 showed very good sensitivity towards C jejuni. C. lari, and C.coli and discrimination against C. upsaliensis and C. fetus.

Example 4: Evaluation of Target Capture and Internal Control Integration

Three Campylobacter 16S rRNA target capture oligonucleotides (TCO) weretested using the SEQ ID NOS:7, 32, 50 & 68 amplification oligonucleotidecombination. The target capture oligos included two Campylobacter TCOsbased upon the sequence of C. jejuni 16S rRNA (SEQ ID NOS:71 & 73) andone wobble TCO (SEQ ID NO:58). An initial heating step is included inthis assay to provide for hybridization of the C. jejuni based TCOs. Thewobble TCO does not require a heating step, however, one was included tokeep the conditions comparable for all three TCOs.

The method of target capture used with Kingfisher 96 is summarized inTable 8. Amplification and Enzyme reagents were reconstituted. A washplate was prepared by filling a KF200 plate with 200 .micro.L/well ofwash solution. An amp plate was prepared by filling another KF200 platewith 100 .micro.L/well of amplification reagent. Both the amp and washplates were covered until used. A sample plate was prepared by adding 50.micro.L TCR/well into a 2-mL, deep-well 96 plate (Axygen). The targetwas titrated from 1E5 copies per reaction to 1E1 copies per reaction in10 .micro.L lysis solution. Specificity was included in the assay bytesting C. fetus and C. upsaliensis at 1E5 copies per reaction. Negativecontrols include lysis reagent only. One ml of lysis solution was addedto each well of the sample plate. With a repeat pipettor, 10 .micro.L oftarget solution was added to the appropriate deep wells. A deep-welltip-comb was placed in the sample plate. The covers for the wash and ampplates were removed. The KF96 protocol was started and all assay plateswere placed on the KF96 instrument. The amp plate was placed in position4, the wash plate in position 3, and the sample (deep-well plate) inposition 1. Position 2 in the KF96 instrument was left empty. Once theplates were loaded, the KF96 instrument began the target capture step.When the KF96 run was completed, the plates were removed. From the ampplate, 30 .micro.L from each well were removed using a multi-channelpipettor and transferred to an MJ 96-well PCR plate.

TABLE 8 Kingfisher 96 Program. Step Step Position Description ActionBeginning Mix End 1 1 Capture Heat 5 min-85.deg.C. Very slow No action 21 Capture Heat 15 min-65.deg.C. Very slow No action 3 2 Cool Heat 30min-25.deg.C. No action No action (table rotated to empty position) 4 1Mix prior to Mix No action 1 min-Very slow Collect beads-collect/collect count 20 Sample 1 5 3 Release to Wash Wash Release 30 sSlow 30 s Slow No action 6 1 Capture Wash Release 30 s Very 30 s VerySlow Collect beads- Sample 2 Slow (mix only) count 20 7 3 Release toWash Release 30 s Slow 30 s Slow Collect beads- Wash 2 count 20 8 4Capture and Wash Release 30 s Slow 30 s Slow No action release into AmpSoln

Results are shown in Table 9, and indicate that SEQ ID NOS:71 & 73 areequivalent to SEQ ID NO:58 for target capture. C. fetus and C.upsaliensis were not detected. C. jejuni, C. coli and C. lari were alldetected at all target levels. SEQ ID NO:73 was selected for furtherCampylobacter detection assays.

TABLE 9 Target Capture Results. Copies of Target Avg TTime TCO Target(Times 10.sup.5) (avg of 4 samples) SEQ ID NO: 71 C. fetus 5 n/a C.jejuni 0 n/a C. jejuni 1 19.5 C. jejuni 2 17.9 C. jejuni 3 15.6 C.jejuni 4 13.3 C. jejuni 5 11.3 C. ups 5 n/a SEQ ID NO: 73 C. fetus 5 n/aC. jejuni 0 n/a C. jejuni 1 19.8 C. jejuni 2 17.4 C. jejuni 3 15.3 C.jejuni 4 13.2 C. jejuni 5 10.9 C. ups 5 n/a SEQ ID NO: 58 C. fetus 5 n/aC. jejuni 0 n/a C. jejuni 1 19.1 C. jejuni 2 16.8 C. jejuni 3 14.8 C.jejuni 4 12.8 C. jejuni 5 11.2 C. ups 5 n/a SEQ ID NO: 71 C. jejuni 510.4 C. lari 0 n/a C. lari 1 17.4 C. lari 2 16.0 C. lari 3 12.7 C. lari4 11.0 C. lari 5 8.4 C. ups 5 21.2* SEQ ID NO: 73 C. jejuni 5 10.0 C.lari 0 n/a C. lari 1 18.2 C. lari 2 15.5 C. lari 3 12.9 C. lari 4 10.3C. lari 5 8.1 C. ups 5 21.6* SEQ ID NO: 58 C. jejuni 5 10.5 C. lari 0n/a C. lari 1 18.1 C. lari 2 15.6 C. lari 3 12.8 C. lari 4 10.2 C. lari5 8.3 C. ups 5 n/a SEQ ID NO: 71 C. jejuni 5 8.3 C. coli 0 n/a C. coli 116.6 C. coli 2 14.4 C. coli 3 12.0 C. coli 4 10.1 C. coli 5 7.5 C. ups 5n/a SEQ ID NO: 73 C. jejuni 5 7.9 C. coli 0 n/a C. coli 1 16.7 C. coli 214.5 C. coli 3 12.4 C. coli 4 10.0 C. coli 5 7.8 C. ups 5 n/a SEQ ID NO:58 C. jejuni 5 8.1 C. coli 0 n/a C. coli 1 15.9 C. coli 2 13.5 C. coli 311.6 C. coli 4 9.2 C. coli 5 7.1 C. ups 5 n/a *1 of 4 samples showed apositive TTime for both test conditions.

Thus, from these assays, it was determined to do further testing onamplification oligo set SEQ ID NOS:7, 32, 50 & 68, using a targetcapture probe SEQ ID NO:73.

Example 5. Sensitivity, Specificity, Interference and Limit of Detection

Sensitivity Testing. Campylobacter jejuni (ATCC 33560) was assayed at1E5 copies per reaction. Lysis buffer was used as the negative control.Twenty positives (1E5 copies of rRNA per assay) were tested using theKingFisher 96 instrument for target capture, an Eppendorf thermomixerfor annealing the primers and for enzyme addition, and the PTI-FP-2®FluoDia plate reader was used for detection. Twenty negative controlreactions were included. The input for target capture was 1 mL, theoutput for target capture was 100 .micro.L, of which 30 .micro.L wasused in the amplification. Positive criterion was set at 1000 RFUs.Nineteen of 20 replicates were to be detected with a >95% positivityrate. If less than 19 replicates were positive after an initial round oftesting, then 40 additional replicates were to be tested. Testing forsensitivity at this stage yielded a 100% rate of positivity and a 10%rate of false positivity. The false positives seen at this stage emergedat very low levels and very late in the amplification process. Becausethe 1000 RFU criterion for positives was not an optimized criterion, thefalse positives are not considered true false positives. Theseconditions were evaluated again in an additional round of testing.Results are shown in Table 10.

TABLE 10 Sensitivity Results Target Amt Total Number Avg Sample ID(copies/rxn) Replicates Positive TTime C. jejuni 1E5 20 20 15.9ATCC33560 No Target-CP0.0 0 20 2 14.8

Specificity Testing. Organisms that were closely related to the targetorganism but were genotypically distinct by rRNA analysis were selectedas negatives. Seven challenge organisms were tested at 1E5 copies perreaction using the KingFisher mL instrument for target capture, anEppendorf thermomixer for annealing the primers and for enzyme addition,and the PTI-FP-2® FluoDia plate reader was used for detection. Challengeorganisms were C. fetus ssp venerealis; C. fetus ssp fetus; C. fecalis;E. coli; C. upsaliensis; S. enteritidis; and H. pylori. The positivecontrol was C. jejuni (ATCC 33560) at 1E4 copies per positive controlreaction. Lysis solution was used as negative control. All organismswere tested in replicate reactions. The positive criterion was set at1000 RFU. Less than or equal to 7 of 140 reaction should be positive.Twenty-eight negative control reactions were included. The input fortarget capture was 1 mL; the output for target capture was 100 .micro.L,of which 30 .micro.L was used in the amplification. Testing forspecificity at this stage yielded about a 98% rate of success indiscriminating against the challenge organisms (3/140 were positive; allE. coli reaction wells) and false positive reactions were detected in 2of 84 negative reaction wells. Thus, false positive rates in the E. coliwells are similar those in the negative control wells. The positivecontrol was 100% positive. Table 11.

TABLE 11 Specificity Results. Copies per Percent Organism ReactionPositive C. fetus ssp venerealis 1E5  0% C. fetus ssp. fetus 1E5  0% C.fecalis 1E5  0% E. coli 1E5 0.5%  C. upsaliensis 1E5  0% S. enteritidis1E5  0% H. pylori 1E5  0% C. jejuni (positive) 1E4 100%  Negative 02.4% 

Interference Testing. The goal of this assay is to detect C. jejuni 16SrRNA at a concentration of 1E3 copies (1 CFU) per sample in the presenceof 0 copies (lysis solution only) or 1E7 copies of nearest neighbororganisms. Seven nearest neighbor challenge organism were tested, whichwere as follows: C. fetus ssp venerealis; C. fetus ssp fetus; C.fecalis; E. coli; C. upsaliensis; S. enteritidis; and H. pylori. C.jejuni was used as the baseline target at 1E3 copies. Assays wereperformed using the KingFisher96 and the PTI reader. All conditions weretested in replicates of 12, with a positive criterion of 1000 RFU.Interference testing showed 100% positivity for detection of C. jejuniin the presence of C. fecalis; E. coli; C. upsaliensis; S. enteritidis;and H. pylori. However, detection of C. jejuni in the presence of C.fetus ssp venerealis; C. fetus ssp fetus yielded 0% positivity and 8.3%positivity, respectively. In a follow-up assay, the interfering C. fetusspecies were similarly tested in combination with C. coli and C. lariand the C. fetus species interfered with detection. The tested C. fetusspecies interfere with detection of C. jejuni, C. coli and C. laritarget nucleic acid using the current amplification oligo combination.

Example 6. Testing an Oligomer Set Using a Variety of T7 Providers

Additional T7 Provider oligomers were prepared and tested. The T7Providers are shown in Table 12, below. Each of the T7 Providers inTable 12 were used in an oligomer combination that included SEQ IDNOS:7, 50 & 68, and one of the T7 Providers. In one condition. theBlocker (SEQ ID NO:50) was not included and so the combination in thisreaction was SEQ ID NOS:7, 26, 68.

TABLE 12 Various T7 Providers SEQ ID NO: Sequence (5′→3′) 32aatttaatacgactcactatagggagaCAGTTGGAAACGACTGCT AATACTCT 84aatttaatacgactcactatagggagaCAGTTGGAAACGACTGCT AAT 85aatttaatacgactcactatagggagaCAGTTGGAAACGACTGC TA 86aatttaatacgactcactatagggagaCAATTGGAAACGACTGCT AATACTCT 87aatttaatacgactcactatagggagaCAGTTGGAAACGACTGCC AACACTCT 88aatttaatacgactcactatagggagaCAGTTGGAAACGACTGCT AACAC 26aatttaatacgactcactatagggagaAAGAGGACAACAGTTGGA AAC

The amplification oligo combinations were screened for sensitivity to C.jejuni, C. coli and C. lari target nucleic acids. A subset of thesecombinations of amplification oligomers was then screened forinterference and cross-reactivity by nearest neighbors. Sensitivityassays were set up as has been generally described herein. C. jejuni(ATCC 33291), C. coli (ATCC 33559) and C. lari (ATCC 35221) were testedat a level of 0, 1E2, 1E3 or 1E4 copies per reaction. Negative controlwas oligoless amplification reagent. Positive selection criterion was atleast 2 of 3 samples having 1000 RFU for the 1E2 reaction and 3 of 3samples having a TTime for RFU 1000 no greater than 20 minutes for the1E4 reaction. Three amplification oligo combinations performed well forsensitivity testing.

Interference Testing. A control amplification oligo combination and thethree amplification oligos combinations that performed best in thesensitivity testing were then further tested for interference using oneof the following inclusive organisms, C. jejuni (ATCC 33291), C. coli(ATCC 33559) or C. lari (ATCC 35221), each combined with one of thefollowing near neighbor challenge organisms, C. fetus ssp fetus, C.fetus ssp venerealis, C. upsaliensis or Archobacter blutzleri. Inclusiveorganisms were tested at 1E5 copies per reaction and challenge organismswere tested at 1E8 copies per reaction. Positive controls were C. jejuni(ATCC 33291) at 1E5 copies per reaction without challenge organism.Negative controls were oligoless amplification reagent. Assays wereperformed using the KingFisher 96 and the PTI reader. Reactions were runin replicates of 12, and positive criterion was 1000 RFU.

All positive controls amplified appropriately and all negative controlshad no false positives. Oligomer combinations with SEQ ID NOS:7, 32, 50& 68, SEQ ID NOS:7, 86, 50 & 68, and to a lesser extent with SEQ IDNOS:7, 84, 50 & 68 showed some interference by C. fetus ssp. fetus andC. fetus ssp. venerealis. No interference was seen with amplificationoligo combination SEQ ID NO:7, 26, 50 & 68. All three of theseamplification oligo combinations were then tested for cross-reactivity.

Cross-Reactivity Testing. Cross-reactivity of the amplification oligoswas tested against 1E8 copies per reaction of C. fetus ssp fetus, C.fetus ssp venerealis, C. upsaliensis and Archobacter blutzleri. Positivecontrols were C. jejuni (ATCC 33291) at 1E5 copies per reaction.Negative controls were oligoless amplification reagent. Reactions wererun in replicates of 4 and positive criterion was 1000 RFU. Positivecontrols amplified appropriately and no false positives were detected.Late, low-slope detection was seen for amplification oligo combinationSEQ ID NO:7, 26, 50 & 68 (average TTime 29.4 minutes), but not withother amplification oligo combinations being tested. Oligomercombination SEQ ID NO:7, 26, 50 & 68 was used for testing a pure culturelysates in buffered-peptone water.

Example 7. Sensitivity, Specificity, Limit of Detection, and AnalyticalTesting for Target Nucleic Acids in Pure Culture Lysates

An oligomer combination was used for testing a pure culture lysates inbuffered-peptone water (BPW). Sample preparation device was notincluded. All positive controls were C. jejuni (ATCC 33560) and allnegative controls were lysis/BPW. All samples are in a 70:30 lysissolution:BPW. The amplification oligos are SEQ ID NOS:7, 26, 50 & 68.The target capture oligo is SEQ ID NO:73.

Sensitivity. Campylobacter jejuni (ATCC 33560), C. coli (ATCC 43478),and C. lari (ATCC 35222) were assayed at 1E4-5E4 copies per reaction.Lysis buffer was used as the negative control. Twenty positives (1E5copies of rRNA per assay) were tested using the KingFisher 96 instrumentfor target capture, an Eppendorf thermomixer for annealing the primersand for enzyme addition, and the PTI-FP-2® FluoDia plate reader was usedfor detection. Twenty negative control reactions were included. Theinput for target capture was 1 mL, the output for target capture was 100.micro.L, of which 30 .micro.L was used in the amplification. Positivecriterion was set at 1000 RFUs. Nineteen of 20 replicates were to bedetected with a >95% positivity rate. If less than 19 replicates werepositive after an initial round of testing, then 40 additionalreplicates were to be tested. Testing for sensitivity at this stageyielded a 100% rate of positivity and a 10% rate of false positivity.

Specificity. Specificity of the amplification oligo combination waschallenged using near neighbor exclusionary organisms. Exclusionaryorganisms were tested at 1E6 copies per reaction using the KingFisher 96and PTI detector. Exclusionary organisms were C. fetus ssp venerealis;C. fetus ssp fetus; C. fecalis; E. coli; C. upsaliensis; S. enteritidis;and H. pylori. The positive control was C. jejuni (ATCC 33560) at 1E5copies per positive control reaction. Lysis solution was used asnegative control. All organisms were tested in replicate reactions. Thepositive criterion was set at 1000 RFU. Specificity testing yielded a100% rate of success in discriminating against the challenge organisms.The E. coli reactions yielded 0 false positives, thus not showing thefalse positive rate that appeared in earlier testing. The positivecontrol was 100% positive. The negative controls showed no falsepositives.

Limit of Detection. C. jejuni (ATCC 33560 and 49351), C. coli (ATCC43478 and 43488), and C. lari (ATCC 35222 and 43675) were tested a levelof 1E3 to 1E4 copies RNA/assay (approximately 1-10 CFU). The actualassay input for each Campylobacter species was 5E3 copies. Targetcapture was performed on the Kingfisher 96 and the detection on the PTIreader. Each species was tested in replicates of 20, from which one 30.micro.L replicate was amplified. The positive criterion for thissection was 1000 RFU, and 19 of 20 replicates must reach this criterionto be considered positive. Note that ATCC 33560 is considered both thepositive control as well as a strain required for testing. It wasincluded as a positive control on the plate where it was not tested at5E3 copies/assay. Results indicated a 100% success rate (20 of 20replicates) for detecting each of these Campylobacter species. Therewere no false positives.

Analytical Testing. The number of organisms tested was expanded toinclude twenty-five organisms for detection and fifteen challengeorganisms. The organisms were tested at 1E5 copies/assay (approximately100 CFU). Testing was performed on the Kingfisher 96 instrument fortarget capture and the PTI reader for detection. All were tested inreplicate reactions of 3, from which one 30 .micro.L replicate wasamplified. For the inclusives, a positive reading in at least 2 of 3replicates and for the exclusives, no more than 1 of 3 replicates shouldbe positive. If these criteria are not met for any organism, testing forthat species/strain will be repeated in replicates of 12. The inclusiveorganisms tested are C. jejuni, C. jejuni ssp doylei, C. coli, and C.lari. The exclusive organisms tested are E. coli, E. coli O157:H7, E.vulnaris, E. hermannii, Enterobacter cloacae, S. enteniditis,Edwardsiella hoshinae, P. mirabilis, Citrobacter brakii, Pseudomonasfluorescens, Shigella flexneri, Aeromonas hydrophila, Arcobacterbutzleri, Campylobacter upsaliensis and Campylobacter fetus ssp fetus.Positive control was C. jejuni (ATCC 33560). All of the inclusiveorganisms yielded 100% positive detection except for two of 16 testedstrains of C. jejuni (ATCC 35920 and 35925). These two strains of C.jejuni yielded 0 of 3 positives. None of the exclusive organisms yieldedany positive results. Positive controls were 100% and there were nofalse positives.

Example 8. Testing an Oligomer Set Using a Variety of Torches

C. jejuni species ATCC 35925 and ATCC 35920 (ATCC, Manassas, Va.) werenot detected in the above analytical testing. Based on sequencing of aregion of interest within the target sequence for these strains of C.jejuni, it was determined that each strain contained a single pointmutation in their nucleotide sequences corresponding to a Torchhybridization region. The location of the point mutation was differentfor each strain. (SEQ ID NOS:89-90).

Sensitivity. During the secondary screening process multiple candidatetorches were considered acceptable based on specificity and sensitivitycondition at that time. For this follow-up experiment, amplificationoligo combinations comprising alternate Torch SEQ ID NOS:67 or originalTorch SEQ ID NO:68 were screened again against C. jejuni ATCC 35925 and35920 at 1E5 copies per reaction. Oligo combinations used were: SEQ IDNOS:7, 32, 50 & 67; SEQ ID NOS:7, 32, 50 & 68; SEQ ID NOS:7, 26, 50 &67; and SEQ ID NOS:7, 26, 50 & 68. C. jejuni (ATCC 33560) was used as apositive control and lysis solution only as the negative control. TheKingfisher 96 instrument was used for the target capture and the PTIreader for the detection. Acceptable alternative amplification oligocombinations will detect of C. jejuni ATCC 35920 and 35925 while stillexcluding nearest neighbors. Positive criterion was set at 1000 RFU.

Control C. jejuni ATCC 33560 was detected with 100% positivity rateusing amplification oligo combinations having Torch SEQ ID NO:67 or SEQID NO:68. The SEQ ID NO:67 assays showed a lower average RFU than didthe SEQ ID NO:68 assays. There were no false positives. For detection ofC. jejuni ATCC 35920, the SEQ ID NO:67 amplification oligo combinationswere 100% positive. The SEQ ID NO:68 combinations could not detect C.jejuni above background. All amplification oligo combinations had asingle false positive result. For detection of C. jejuni ATCC 35925,both the SEQ ID NO:67 and the SEQ ID NO:68 amplification oligocombinations were 100% positive; however SEQ ID NO:67 resulted in ahigher average RFU than did SEQ ID NO:68. Amplification oligocombinations comprising SEQ ID NO:67 were then tested for discriminationagainst exclusionary organisms.

Selectivity. Two analytical tests for exclusive species were performedusing amplification oligo combinations comprising SEQ ID NO:67 andtwelve exclusionary organisms in a first assay followed by threeexclusionary organisms in a second assay. The second assay was performedto retest false positives identified in the first. The exclusionaryorganisms for the first assay were provided at 1E5 copies per reactionand included A. butzleri, C. fecalis, C. fetus ssp. fetus, C. fetus ssp.fetus, C. fetus ssp. venerealis, C. upsaliensis, C. upsaliensis, E.coli, E. faecalis, E. faecium, P. fluorenscens and S. enteritidis.Positive control was 1E5 copies per reaction of C. jejuni ATCC 33560 andnegative control was lysis solution. All reactions were in replicates ofthree. No more than 1 of 3 reactions should be positive for theexclusionary organisms tested. All exclusionary organisms were 100%negative except for C. upsaliensis, E. faecalis and S. enteritidis; eachof which had 1 of 3 false positive reactions.

Though 1 of 3 false positives meets the criteria for a negativereaction, a second assay was performed on the each of the speciesshowing a single false positive result. In this assay the exclusionaryorganisms were provided at 1E5 copies per reaction and included C.upsaliensis, E. faecalis and S. enteritidis. Positive control was 1E5copies per reaction of C. jejuni ATCC 33560 and negative control waslysis solution. All reactions were in replicates of eight. No more than1 of 8 reactions should be positive for the exclusionary organismstested. All exclusionary organisms were negative for 7 of 8 reactions.Amplification oligo reactions comprising Torch SEQ ID NO:67 overcome theloss of sensitivity for C. jejuni ATCC 35920 and 35925 that was seenabove with SEQ ID NO:68.

Example 9. Poultry Rinsate Testing

To test the functionality of the prototype Campylobacter assay, twentypoultry rinsates were prepared. Rinsates were obtained by placing awhole defeathered chicken carcass (approximately 2-5 pounds) in a largesealable plastic bag with 400 mL of buffered peptone water (BPW). TheBPW was distributed over the chicken by shaking the bag and using manualexertion on the outside of the bag. All reactions were in replicates offour. Direct detection of Campylobacter was performed by assaying 500.micro.L of rinsate using amplification oligo combination SEQ ID NOS:7,26, 50 & 67 and the target capture oligo SEQ ID NO:73. Campylobacter andinternal control amplification oligos were used at the followingconcentrations: 5 pM T7 Provider, 0.5 pM Blocker, 5 pM Primer and 8 pMTorch. Rinsates were also assayed using a culture method substantiallysimilar to the USDA recommended culture methods for compare.Campylobacter jejuni ATCC 33291 was the positive control at 1E5 copiesper reaction. Lysis buffer was used as the negative control. Assaysusing the KingFisher 96 instrument for target capture, an Eppendorfthermomixer for annealing the primers and for enzyme addition, and thePTI-FP-2® FluoDia plate reader for detection. The input for targetcapture was 500 .micro.L, the output for target capture was 100.micro.L, of which 30 .micro.L was used in the amplification. Positivecriterion was set at 1000 RFUs. Amplification of Campylobacter was foundfor all rinsates with a mean and median TTime of 18.7 minutes. No falsepositives were detected. Table 13. Using the USDA culture method therewas no detection of Campylobacter species in any of these rinsates.

TABLE 13 Rinsate Average TTimes Avg Sample ID TTime Rinsate #1 19.4Rinsate #2 22.6 Rinsate #3 20.8 Rinsate #4 16.4 Rinsate #5 22.4 Rinsate#6 20.9 Rinsate #7 18.4 Rinsate #8 16.6 Rinsate #9 18.6 Rinsate #10 16.7Rinsate #11 18.8 Rinsate #12 17.3 Rinsate #13 19.6 Rinsate #14 18.9Rinsate #15 15.6 Rinsate #16 18.2 Rinsate #17 16.7 Rinsate #18 19.7Rinsate #19 17.0 Rinsate #20 18.9 Non Template 28.1 Positive Control19.0

In summary, real-time TMA technology was suitable for rapid, highlysensitive detection of food-borne pathogens. The assay had a sensitivityof 5E3 target nucleic acid copies/assay (approximately 10 CFU) for thedesired species, C. jejuni, C. coli and C. lari, while excluding variousnearest neighbors and potentially co-contaminating flora at 1E7 targetnucleic acids copies/assay (approximately 10,000 CFU). The datademonstrated a rapid test format that allowed screening of food samplesfor Campylobacter within a single 8-hour work shift.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

1-58. (canceled)
 59. A method for specifically detecting one or more ofa Campylobacter jejuni, Campylobacter coli and Campylobacter lari targetnucleic acid in a sample comprising the steps of: (a) obtaining a samplesuspected of containing at least one of a Campylobacter jejuni,Campylobacter coli and Campylobacter lari target nucleic acid; (b)contacting said sample with a combination of a first amplificationoligomer and a second amplification oligomer, wherein the firstamplification oligomer comprises a contiguous target hybridizingsequence 15 to 45 nucleotides in length and configured to hybridize to asequence in a region of a Campylobacter 16S rRNA gene corresponding tonucleotides 83 to 150 of SEQ ID NO:91 and wherein the secondamplification oligomer comprises a contiguous target hybridizingsequence 15 to 45 nucleotides in length and configured to hybridize to asequence in a region of the a Campylobacter 16S rRNA gene correspondingto nucleotides 170 to 226 of SEQ ID NO:91; (c) performing an in vitronucleic acid amplification reaction wherein any of a C. jejuni targetnucleic acid, a C. coli target nucleic acid and a C. lari target nucleicacid present in the sample is used as a template for generating anamplification product; and (d) performing a nucleic acid detectionreaction that detects the amplification product to determine whether aCampylobacter target nucleic acid was present in the sample.
 60. Themethod of claim 59, wherein the combination of the first amplificationoligomer and the second amplification oligomer comprises targethybridizing sequences selected from the group consisting of: (i) SEQ IDNO:19 and SEQ ID NO:4; (ii) SEQ ID NO:19 and SEQ ID NO:7; (iii) SEQ IDNO:19 and SEQ ID NO:12; (iv) SEQ ID NO:33 and SEQ ID NO:7; (v) SEQ IDNO:33 and SEQ ID NO:12; (vi) SEQ ID NO:49 and SEQ ID NO:4; (vii) SEQ IDNO:49 and SEQ ID NO:7; (viii) SEQ ID NO:49 and SEQ ID NO:12; (ix) SEQ IDNO:29 and SEQ ID NO:7; (x) SEQ ID NO:31 and SEQ ID NO:7; (xi) SEQ IDNO:25 and SEQ ID NO:7; (xii) SEQ ID NO:25 and SEQ ID NO:12; (xiii) SEQID NO:33 and SEQ ID NO:4; and (xiv) SEQ ID NO:33 and SEQ ID NO:7. 61.The method of claim 59, wherein the first amplification oligomer targethybridizing sequence comprises a 5′ promoter sequence.
 62. The method ofclaim 61, wherein the first amplification oligomer comprises a nucleicacid sequence consisting of SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:28;SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:48.
 63. The method of claim 59,wherein step (b) further comprises contacting the sample with a blockeroligomer.
 64. The method of claim 63, wherein the blocker oligomercomprises a nucleic acid sequence consisting of SEQ ID NO:50, SEQ IDNO:53, SEQ ID NO:56 or SEQ ID NO:57.
 65. The method of claim 59, whereinthe detection reaction is a real-time detection reaction.
 66. The methodof claim 59, wherein the detection reaction of step (d) comprisescontacting the amplification product with a detection probe oligomerconfigured to hybridize to a portion of the amplification product. 67.The method of claim 66, wherein the detection probe oligomer comprisinga nucleic acid sequence consisting of SEQ ID NO:63, SEQ ID NO:65, SEQ IDNO:66, SEQ ID NO:67, or SEQ ID NO:68.
 68. The method of claim 59,wherein the sample further contains nucleic acid from one or morebacteria closely related to C. jejuni; C. coli, or C. lari and thetarget nucleic acid is specifically detected in the sample.
 69. Themethod of claim 68, wherein said one or more bacteria is at least oneof: C. fetus, ssp. fetus, C. fetus ssp venerealis, C upsaliensis, E.coli, C. fecalis, S. enteridis, H. pylori, E. vulnaris, E. hermannii,Enterobacter cloacae, Edwardsiella hoshinae, P. miribalis, Citrobacterbrakii, Pseudomonas fluorescens, Shigella flexneri, Aeromonashydrophila, A. butzleri, C. fetus, ssp. fetus, C. fetus ssp venerealis,and C. upsaliensis.
 70. The method of claim 66, comprising a combinationof first amplification oligomer, second amplification oligomer anddetection probe oligomer comprising nucleic acid sequences consistingof: (1) SEQ ID NO:18, SEQ ID NO:4, and SEQ ID NO:63; (2) SEQ ID NO:18,SEQ ID NO:7, and SEQ ID NO:63; (3) SEQ ID NO:18, SEQ ID NO:12, and SEQID NO:63; (4) SEQ ID NO:18, SEQ ID NO:7, and SEQ ID NO:68; (5) SEQ IDNO:18, SEQ ID NO:12, and SEQ ID NO:68; (6) SEQ ID NO:32, SEQ ID NO:4,and SEQ ID NO:63 (7) SEQ ID NO:32, SEQ ID NO:7, and SEQ ID NO:63; (8)SEQ ID NO:32, SEQ ID NO:12, and SEQ ID NO:63; (9) SEQ ID NO:32, SEQ IDNO:7, and SEQ ID NO:65; (10) SEQ ID NO:32, SEQ ID NO:7, and SEQ IDNO:66; (11) SEQ ID NO:32, SEQ ID NO:7, and SEQ ID NO:67; (12) SEQ IDNO:32, SEQ ID NO:7, and SEQ ID NO:68; (13) SEQ ID NO:32, SEQ ID NO:12,and SEQ ID NO:68; (14) SEQ ID NO:48, SEQ ID NO:4, and SEQ ID NO:63; (15)SEQ ID NO:48, SEQ ID NO:7, and SEQ ID NO:63; (16) SEQ ID NO:48, SEQ IDNO:12, and SEQ ID NO:63; (17) SEQ ID NO:48, SEQ ID NO:7, and SEQ IDNO:68; (18) SEQ ID NO:48, SEQ ID NO:12, and SEQ ID NO:68; (19) SEQ IDNO:28, SEQ ID NO:7, and SEQ ID NO:68; (20) SEQ ID NO:30, SEQ ID NO:7 andSEQ ID NO:68; (21) SEQ ID NO:24, SEQ ID NO:7, and SEQ ID NO:68; and (22)SEQ ID NO:24, SEQ ID NO:12, and SEQ ID NO:68.
 71. The method of claim70, wherein step (b) further comprises contacting the sample with ablocker oligomer.
 72. The method of claim 71, wherein the blockeroligomer comprises a nucleic acid sequence consisting of SEQ ID NO:50,SEQ ID NO:53, SEQ ID NO:56 or SEQ ID NO:57.
 73. The method of claim 72,wherein the detection probe oligomer comprises a detectable label. 74.An amplification reagent comprising: a buffer, inorganic salt, dNTPs,and a combination of first amplification oligomer and a secondamplification oligomer, wherein the first amplification oligomercomprises a contiguous target hybridizing sequence 15 to 45 nucleotidesin length and configured to hybridize to a sequence in a region of aCampylobacter 16S rRNA gene corresponding to nucleotides 83 to 150 ofSEQ ID NO:91 and wherein the second amplification oligomer comprises acontiguous target hybridizing sequence 15 to 45 nucleotides in lengthand configured to hybridize to a sequence in a region of the aCampylobacter 16S rRNA gene corresponding to nucleotides 170 to 226 ofSEQ ID NO:91.
 75. The amplification reagent of claim 74, wherein thefirst amplification oligomer and the second amplification oligomercomprise target hybridizing sequences selected from the group consistingof: (i) SEQ ID NO:19 and SEQ ID NO:4; (ii) SEQ ID NO:19 and SEQ ID NO:7;(iii) SEQ ID NO:19 and SEQ ID NO:12; (iv) SEQ ID NO:33 and SEQ ID NO:7;(v) SEQ ID NO:33 and SEQ ID NO:12; (vi) SEQ ID NO:49 and SEQ ID NO:4;(vii) SEQ ID NO:49 and SEQ ID NO:7; (viii) SEQ ID NO:49 and SEQ IDNO:12; (ix) SEQ ID NO:29 and SEQ ID NO:7; (x) SEQ ID NO:31 and SEQ IDNO:7; (xi) SEQ ID NO:25 and SEQ ID NO:7; (xii) SEQ ID NO:25 and SEQ IDNO:12; (xiii) SEQ ID NO:33 and SEQ ID NO:4; and (xiv) SEQ ID NO:33 andSEQ ID NO:7.
 76. The amplification reagent of claim 75, wherein thewherein the first amplification oligomer target hybridizing sequencecomprises a 5′ promoter sequence.
 77. The amplification reagent of claim75, wherein the reagent comprises a blocker oligomer.
 78. Theamplification reagent of claim 75, wherein the reagent comprises adetection probe oligomer.
 79. The amplification reagent of claim 78,wherein the first amplification oligomer, the second amplificationoligomer and the detection probe oligomer comprise nucleic acidsequences selected from the group consisting of: (1) SEQ ID NO:18, SEQID NO:4, and SEQ ID NO:63; (2) SEQ ID NO:18, SEQ ID NO:7, and SEQ IDNO:63; (3) SEQ ID NO:18, SEQ ID NO:12, and SEQ ID NO:63; (4) SEQ IDNO:18, SEQ ID NO:7, and SEQ ID NO:68; (5) SEQ ID NO:18, SEQ ID NO:12,and SEQ ID NO:68; (6) SEQ ID NO:32, SEQ ID NO:4, and SEQ ID NO:63 (7)SEQ ID NO:32, SEQ ID NO:7, and SEQ ID NO:63; (8) SEQ ID NO:32, SEQ IDNO:12, and SEQ ID NO:63; (9) SEQ ID NO:32, SEQ ID NO:7, and SEQ IDNO:65; (10) SEQ ID NO:32, SEQ ID NO:7, and SEQ ID NO:66; (11) SEQ IDNO:32, SEQ ID NO:7, and SEQ ID NO:67; (12) SEQ ID NO:32, SEQ ID NO:7,and SEQ ID NO:68; (13) SEQ ID NO:32, SEQ ID NO:12, and SEQ ID NO:68;(14) SEQ ID NO:48, SEQ ID NO:4, and SEQ ID NO:63; (15) SEQ ID NO:48, SEQID NO:7, and SEQ ID NO:63; (16) SEQ ID NO:48, SEQ ID NO:12, and SEQ IDNO:63; (17) SEQ ID NO:48, SEQ ID NO:7, and SEQ ID NO:68; (18) SEQ IDNO:48, SEQ ID NO:12, and SEQ ID NO:68; (19) SEQ ID NO:28, SEQ ID NO:7,and SEQ ID NO:68; (20) SEQ ID NO:30, SEQ ID NO:7 and SEQ ID NO:68; (21)SEQ ID NO:24, SEQ ID NO:7, and SEQ ID NO:68; and (22) SEQ ID NO:24, SEQID NO:12, and SEQ ID NO:68; and wherein the detection probe oligomercomprises a detectable label.